_Amhives

Arch Virol (1996) 141:1961-1977

Vi rology
© Springer-Verlag 1996
Printed in Austria

Molecular detection of bean common mosaic and bean common

mosaic necrosis potyviruses and pathogroups

L. Xu 1 and R. O. H a m p t o n 2

1 Department of Botany and Plant Pathology, 2 U.S. Department of Agriculture,

Agricultural Research Service, Department of Botany and Plant Pathology,
Oregon State University, Corvallis, Oregon, U.S.A.

Accepted May 15, 1996

Summary. Partial nucleotide sequences of selected isolates of bean common

mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) were
determined. Based on these sequences and previously published sequence data,
a reverse transcription, polymerase chain reaction (RT-PCR) in combination
with restriction endonuclease analyses, was developed for molecular detection of
BCMV, BCMNV and some viral pathogroups (PG). Specific detection of the two
viruses was accomplished by constructing two virus-specific primer pairs that
amplified a PCR product specific for each virus. By application of RT-PCR, four
BCMV-PG-V isolates were differentiated from isolates of BCMV pathogroups I,
II, IV and VII. Distinction of two BCMNV pathogroups (PG-III and PG-VI)
was achieved by restriction enzyme XbaI digestion of BCMNV PCR products.
However, no combination of tested restriction enzymes distinguished all five
recognized BCMV pathogroups. A primer pair Dts/Unyi 5 proved to be specific
for BCMV pathogroup PG-V. Thus, by a combination of RT-PCR and restric-
tion enzyme analyses, it was possible to differentiate both viruses, two pathog-
roups of BCMNV, and one pathogroup of BCMV from the others.

Introduction
Bean common mosaic necrosis potyvirus (BCMNV) was recently differentiated
from bean common mosaic potyvirus (BCMV) [11, 12-] and the new classifica-
tion was approved [13-]. The viruses were previously distinguished on the basis of
reactions of differential bean cultivars [2, 3, t6] and by serology [12]; however,
both induced similar symptoms in numerous bean (PhaseoIus vulgaris L.)
genotypes including mosaic, leaf distortion, stunting, and lethal necrosis [2, 3, 12,
16]. As potyviruses, their single stranded positive sense RNA genomes are
presumed to encode a large polyprotein that is subsequently processed. To date,
only capsid protein gene sequences of the two viruses have been published [5].
1962 L. Xu and R. O. Hampton

The efficacious classification of BCMV strains, hereafter called pathotypes,

and the definition of P. vulgaris pathotype-resistance genes by Drijfhout [3]
encompassed pathotypes of both BCMV and BCMNV, which facilitated
unprecedented success in BCMV resistance breeding. A significant biological
distinction between the two viruses is that BCMNV infects the P. vulgaris
cuttivars of the I, I genotype, causing lethal temperature-independent systemic
necrosis, as opposed to temperature-dependent systemic necrosis induced by
BCMV pathogenicity groups, hereafter called pathogroup IVb and Vb.
Seven "BCMV" pathogenicity groups were differentiated by Drijfhout [3],
based on the systemic infectability of selected bean cultivars. Thus, relative to
current classification, Drijfhout pathogroups III and VI now comprise BCMNV,
and pathogroups I, II, IV, V, and VII comprise BCMV [13].
Several diagnostic approaches have been applied to BCMV and BCMNV
detection and differentiation, including polyclonal antisera to intact
virions/capsid protein [9] and to N-terminal amino acids of viral capsid protein
[4], monoclonal antibodies [12] and HPLC of partially digested capsid protein
[11]. The two viruses were clearly distinguished by most of these approaches.
However, differentiation of pathogroups was achievable by none. Pathogroups
still must be defined by symptomatic reactions of selected P. vulgaris genotypes,
which requires much effort, large amounts of space and time, and sometimes
yields equivocal results [3].
Accordingly, applications of current molecular approaches were viewed as
an attractive alternative approach. For instance, the recent amplification of
virus-specific nucleotide sequences by reverse transcription and polymerase
chain reaction (RT-PCR) was used for specific detection and differentiation of
pea seedborne mosaic potyvirus pathotypes [7, 8]. Likewise, three tobamovirus
pathotypes infecting L-resistant pepper genotypes were differentiated, using
RT-PCR and restriction-enzyme analysis [15]. Such approaches were not yet
reported for the detection of BCMV and BCMNV.
This paper reports the development and application of RT-PCR and restric-
tion-enzyme analyses for differentiation of BCMV, BCMNV and some of their
pathogroups.

Materials and methods

Virus isolates, purification and RNA extraction
The viral isolates investigated and their identities and sources are shown in Table 1. The
pathogroup identity of each isolate was confirmed or determined by differential bean
genotypes, as described by Drijfhout [3]. Virus purification and RNA extraction were
performed by standardized methods, as previously described [6-8].

Primer design
Initially, a pair of broad-spectrum primers Db/Udg (Table 2), was designed to amplify an
unknown sequence region from both BCMV and BCMNV genomes. The degenerate primer
Udg was selected from conserved sequences in the polymerase coding region of several
 Molecular detection of BCMV and BCMNV 1963

Table 1. Viral isolates, their identities and sources

Virus" Isolate Pathogroup b Source ~

BCMV Type I MJS

TS I ROH
NL1 I MJS
Iran I MJS
PV25 II MJS
NL7 II MJS
Florida IV MJS
Western IV MJS
NL6 IV MJS
tD123 IV MJS
NW63-108 a IV MJS
NY15 V ROH
NY15 (Prov) V MJS
NY15 (Dean) V MJS
NY15 (Zau) V MJS
Mex VII MJS
NL4 VII MJS
BCMNV NL8 (Drij) III DRIJ
NL8 (Bash) III ROH
TNt VI MJS
NL3 VI MJS
NLS VI MJS

"BCMV Bean common mosaic potyvirus; B C M N V bean common

mosaic necrosis potyvirus. BCMNV was formerly considered to be
a temperature-independent necrosis inducing variant of BCMV
bSee Drijfhout [3]
~MJS M. J. Silbernagel, USDA-ARS, Prosser, WA; ROH R. O.
Hampton, USDA-ARS, Corvallis, OR; DRIJ E. Drijfhout, Wageningen,
The Netherlands. Isolates had either originated with E. Drijfhout or
were field-derived by others and proven true-to-type on standardized
bean-cultivar differentiais
d Original pathotype description of isolate NW63-t08 (MJS, un-
publ.) was PG-VII

potyviruses (GenEMBL Database Accession Nos. x676673, M96425, M92280, D10930,

D00441, $42280, M 15239), and primer Db was selected from the capsid protein coding region
of certain BCMV isolates (4, GenEMBL Database Accession Nos. L 11890, L21767, L 15332,
L12740, L 19539, Z17203). The region amplified by Db/Udg relative to previously published
potyviral genomes and BCMV sequence data is shown in Fig. la, b and c. Two restriction
sites, XbaI and PstI, were incorporated into the two primers to facilitate subsequent cloning
and sequencing processes.
 4~

Table 2. Primer descriptions

Primer ~ Sequence b Target Position ~ P C R product

size

Db 5 ' - A C T C G C C C T G C A G A G C A T T G T A C C - 3 'd BCMV + BCMNV 757-780 980

Udg 5'-GYGGNCARCCWTCTAGAGTKGTKGAYA 1698-1737
AYACHYTOATGG-3'
~z
Dbcmv 5'-ACCACGCTGCAGCTAAAGAGAACA-3' BCMV 162-185 1 456
Ubcmv 5 ' - A A T C T A G A T G A T A T C A T A C T C T C T A - 3' 1594-1618
Dnl3 5'-GAATTGAAAGCGTACTATCTAATACAG-3' BCMNV 164-190 922 ©
Unl3 5'-CAGCTTGAATTTGATTCTGATGATGAGGTG-Y 1057 1086
Dts 5'-TGCAGTGTGCCTTTCAGTATTCTCGCT-3' BCMV-PG-V 305-331 1 164
Unyl5 5'-TATACAAGTGGACGGAG-3' 1453-1469
©

~D Downstream primer; U upstream primer

b Letter code for nucleotide at degenerate position: Y = C, T; R = A, G; W = A, T; K = G, T; H = A, C, T; O = G, C, T; N = A, C, G, T
Positions of primers coordinate in nucleotides from 3' end
d Inserted restriction sites are underlined. XbaI site: T C T A G A ; PstI site: C T G C A G
 Molecular detection of BCMV and BCMNV 1965

pl HC-Pro P3 CI 6K Nla NIb CP 3'

t i I .... fl I i t~y\A

\
3'(1196)

b. 5'(1) 3'(956)

Uicmv~ ..~crnv
C,

Unyl5 Dts.

Fig. 1. A consensus potyviral genome map. a Region of the BCMV genome for which RNA
nucleotides were previously published; b region of the BCMV genome for which sequence
was determined in this study; e positions of constructed primer pairs, based on the combined
(overlapped) sequences of a and b (see Table 2). Parenthetical nucleotide numbers indicate
lengths of individual and combined sequences, not nucleotide numbers assigned to the
BCMV genome

After partial sequences were determined for selected isolates of BCMV and BCMNV (see
below), two virus specific-primer pairs (Dbcmv/Ubcmv, Dnl3/Unl3) and one pathogroup
specific-primer pair (Dts/Uny 15) were designed from sequences developed in our lab, as well
as from previously published sequences. The primer sequence, the expected PCR product
size, and genome positions respectively are shown in Table 2 and Fig. lc. Primer pair
Dts/Uny 15 should have been specific for BCMV-PG-V. In experimental trials, however, the
sequence in primer Dts was found also to be conserved in isolates of other pathogroups, and
only primer Unyl 5 proved to be PG-V-specific. The sequence of primer Uny15 differed from
sequences of non-NY-15 isolates by only a single 3'-end nucleotide.

Reverse transcription and polymerase chain reaction ( R T-PC R )

Reverse transcription was performed in a 20 pl reaction mixture containing 50 mM MgzC1,
50raM KC1, 10mM Tris-HC1 (pH 8.3), l m M each dNTP, 20U RNase Inhibitor, 50U
M-MLV reverse transcriptase, 100 pM downstream primer and less than 1.0 gg total RNA.
The mixture was overlaid with 75 pl mineral oil and incubated at 42 °C for 1 h, and then
heated to 99 °C for 5 min to inactive the reverse transcriptase.
PCR was performed in the same tube, in a 100-gl reaction mixture containing
50raM KC1, 2raM Mg2C1, 10mM Tris-HC1 (pH 8.3), 0.2mM each dNTP, 100pM of
upstream primer and 2.5 U Taq DNA polymerase. Thirty-five reaction cycles were per-
formed: 1 rain intervals at 37 °C for annealing, 1 min at 72 °C for extension, and 1 rain at 94 °C
for melting.
Aliquots (8gl) of PCR amplification product were analyzed by electrophoresis in 1.2%
agarose gels stained with 0.51ag/ml ethidium bromide.
1966 L. Xu and R. O. Hampton

Amplification, cloning and sequencing of R T-PCR products

Five BCMV isolates (Ts, NL7, NY15, Florida and Mex) and BCMNV isolate (NL3)
representing six distinct pathotypes (pathogroup representatives) were selected for amplifica-
tion with primer pair Db/Udg. The amplified PCR products of predicted size were isolated
from gels by Prep-A-Gene DNA purification kit (Bio-Rad Laboratories, 3300 Regatta
Boulevard, Richmond, CA 94804), and subsequently cloned into the XbaI-PstI sites of
plasmid pUC118 and pUC119, as described by Maniatis et al. [10]. Clones shown to have the
correct insert were sequenced from both directions by the Service Laboratory of the Center
for Gene Research, Oregon State University, using an ABI Model 373A DNA Sequencer
with the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Applied
Biosystems, Inc. 850 Lincoln Center Drive, Foster City, CA 94404). An internal primer was
also constructed for sequencing the central region of the PCR products.
Sequence assembly and alignment was accomplished using the GCG (Program Manual
for the GCG Package, Version 7, April 1991, 575 Science Drive, Madison, Wisconsin, USA)
and the IG (IntelliGenetics, Inc.) software package.
Sequences were recorded in the GenBank Nucleotide Database under accession numbers
US5315, US5316, US5317, US5318, US5319, and US5320.

RestricHon endonuclease analysis

PCR products generated from the two virus-specific primer pairs (Dbcmv/Ubcmv,
Dnl3/Unl3) were subjected to restriction endonuclease analysis. Four restriction enzymes,
XbaI, EcoRI, BamHI and EcoRV, were selected from sequence analyses and used. Restric-
tion enzyme digestion was performed overnight at 37 °C in 8 gl reaction mixtures containing
5 gl PCR product solution, 0.8 g110x restriction enzyme buffer, 2.2 gl water and 2-5 units of
restriction enzyme. The digestion products were resolved in 1.2% agarose gels by elec-
trophoresis.

Results
Sequence analysis of RT-PCR products
Predicted size PCR products (approx. 980 bp) were amplified from all six isolates
(TS, NY15, NL7, Florida, Mex and NL3) with primer pair Db/Udg. Nucleotide
sequences of the respective PCR products are presented in Fig. 2. Sequences
from the 5 BCMV isolates were highly conserved both in length (956 bp) and in
homology (86% to 97%). B C M N V isolate NL3 was less conserved relative to
BCMV, with 879 bp and 65% to 67% homology. Comparisons of the putative
amino acid sequence (Fig. 3) revealed that the most diverse region between
BCMNV-NL3 and BCMV isolates (i.e., pathogroup representatives) was in the
N-terminal regions of the capsid proteins (CP). This region of NL3 comprised 26
amino acids compared to 52 amino acids of BCMV isolates, with 46% to 58%
homology to BCMV isolates. The conserved motif DAG, previously reported to
be required for aphid transmission [1], occurred in the CP N-terminus of all
isolates except BCMV-NY15.

RT-PCR amplification with specific primer pairs

Twenty-two isolates of BCMV and B C M N V (Table 1) were subjected to
RT-PCR amplification with 3 specific primer pairs (Table 2, excluding Db/Udg)
 Molecular detection of BCMV and BCMNV 1967

1 50
florida AGAGTGGTTG ATAATTCCCT TATGGTCGTG ATGTCAG~PT ACTACTCGTG
:II,,,,,
..... I I I~,,,,I~ .....
,,,,,IIII ,,,'"III~'",~, II:~I~I '~',,,
mex AGAGTGGTGG ACAATTCCCT GATGGTCGTG ATGTCAGTTT ACTACTCGTG

n17 AGAGTGGTGG ATAACTCCTT TATGGTTGTG ATGTCA~ AC~fACTCGTG

nyl5 AGAGT~G~ AC~CA~2 GATGGTAGTA A'I~TCAG~'~T A~I~2A~TCA~

ts AGAG~'~TGG A~k~I~CA'fT TATGGTTGTA A ~ T ~ G T ' I ~ A~TA~"fCA~

nl3 AGAGTGGTGG A C ~ C A ~ r GATGG~I~GTC A ' P I ~ C ~ ' I ~ T ACTA~rCA'I~

51 I00
florida TCAC.~'IW GG~2GGAGCG A~GAGGACAT A C ~ G A G C G T ~T"I'~I'~

~ex TCACAAGGTT GGCTGGAGCG ATGATGACAT ACAAGAGCGT CTGGTTTTCT

~17 TCACAAGGTG GGCTGGAGTG ATGAA~ACAT ACAAGAGCGT CTGGTTTTCT

nyl5 CCATAAGGTG GG~2GGAGTG ATGAGGACAT ACAAGAACGT TTGGTCTTCT

ts TCACAAGGTG GGGTGGAGCG ATGAGGACAT ACAAGAGCGT TTGGTTTTCT

n13 ~T~GAG GG~GA~ Aq~ATGATAT T C A G ~ A A C ~ ~TAG~"~I~TCT

I01 150
florida ~IWGC/~'I~G AGATGATATC ATAC~CTCTA T A C . ~ G / ~ G T GGA~I~TG'I~G
i i ~ l l l l l l I I ~ l l l l { I I l l l l l l l l [ ll~llll(l I ~ l l l l [ l l l

mex TCGC~'IN3G AGATGATATC A T A C T C I ~ ' f A TAC/~G~GT GGA~'TGTGG

n17 TTGCAAATGG AGATGATATC ATACTCTCTA TACAAGAAGT GGACTTGTGG

nyl5 TTGCGAATGG AGACGATATC ATACTCWCTA TACAAGAAG~ GGACTTGTGG

ts 'I~GC~DGG AGA'I~AC~TC ATACTCTCTA T A C / ~ G A / ~ C GGACTTGTGG

I ~I II II ,,,,''"I Ill IIII~ 1 I III ,," III ,,"III
hi3 TCGCCAACGG TGATGATATC ATACTAGCAG TTCAAAAAGA GGATGTGTGG

151 200
florida GTTCTCGACA CATTTGCTGC ATCATTCAAA GAGCTGGGTT TAAACTACAA

mex GTTCTTGACA CATTTGCTGC ATCATTCAAA GAGCTGGGTT TGAATTACAA

hi7 GTTC~TGACA CATTTGCTGC ATCATTTAAA GAGCTGGGTT TGAACTATAA

nyl5 GTACTTGATA CATTTGCTGC ATCGTTCAAA GAACTGGGTT TGAACTACAA

:I I:~II I ,,"II ~:: I III II I I II ,,,,I I ~I,,,,IIII
ts GTTCTTGACA CATTCGCTAC ATCATTTAGA GAGCTGGGAT TGAACTACAA
' I I .i [ . . .~1. . .i.1 . i ii I ''~
iii I II'",,, II II 1.....
141[ 1
~13 TTATATAACA CACTCAGCAA TTCCTTTAAA GAGCTCGGTC TGAACTATGA

201 250
florida CTTCGATGAG AGAACAAGGA AGAGAGAAGA CCTCTGGTTT ATGTCACACT

mex CTTTGATGAG AGAACAAGGA AGAGAGAAGA CCTCTGGTTT ATGTCACACT

" :lllIl '"III .... IIIII",,~,, ,~,l:llll II~,,,LIII
n17 TTTCGATGAG AGAACAAGGA AGAGAGAAGA TCTCTGGTTT ATGTCACACT

nyl5 CTTTGATGAA AGAACAAGAA AGAGAGAGGA TCTCTGGTTC ATGTCACACT

..... I~I ,," IIII 1
ts TTTTGATGAG AGGACAAAGA AGAGAGAGGA CCTCTGGTTC ATGTCGCACT

nl3 TTTTNCAGAA CAAACTACAA AGCGTGAGGN GCTATGGTTT ATGTCACATC

251 30O
florida GTG~TATACA GGTGGATGGA A T T T A T A T T C C A A A A T T G G A G C C G G A G C G T
.... I ' ' " I :Ill .... II II: .... Ill
mex GTGCTATACA GGTGGATGGA ATTTATATTC CAAAATTGGA GCCGGAGCGT

nl7 GTGCTATACA GGTGGACGGA ATTTATA~C CAAAATTGGA GCCAGAGCGT

nyl5 GTGCTATACA G~TGGACGGA GTTTACATTC CAAAACTGGA ACCAGAGCGA

ts GTGCAATCGA AGTGGATGGA ATTTACATTC CAAAGCTAGA GCCAGAGCGT

nl3 AAGCAATGCT AATTGATGAT ATATATATAC CAAAGCTTGA GCAAGAAAGA

301 35O
florida GTGGTL~i?CGA T~DI~AG~TG GGACAGGAGC AAGGAAATGA TGCACAGAAC

mex GTGGTCTCGA T T T T A G ~ T G GGACAGGAGC AAGGAAATGA TGCATAGAAC

illlllllf~ i[(1t ~111 i ~t~*
n17 GTGGTCTCGA ' I " I ' T T A G ~ T G G G A C A G G A G C A A G G A G A T G A T G C A T A G A C C
i' 'i I I I I I I ,,'' [ I I ,,'' I:III,,,,, II IIII:II IIII,,,, .... I
nyl5 GTTGTTTCGA TTCTGGAGTG GGACAGGAGC AAAGAGATGA TGCATAGAAC
II I " I I I I ''" II:" I:I'I''",
,,,, II II ILII ~Ill, I I:
ts GTGGTTTCGA TTCTAGAGTG GGACAGGAGG AAGGAAATGA TGCACCGGAC

nl3 ATCGTATCCA ~AGAATG GGATCGAAGC AAAGAACTCA TGCATAGAAC

Fig. 2 (co~einued)
1968 L. Xu and R. O. Hampton

351 400
florida TGAAGCAATC TGTGCAGCAA TGATAGAAGC GTGGGGATAT CCTGAGCTAC

mex TGAAGCAATC TGTGCAGCAATGATAGAAGC GTGGGGATAT CCTGAGCTGC

n17 TGAAGCAATC TGTGCAGCAA TGATAGAAGC GTGGGGGTAT CCTGAGCTAC

1111 ~b illll Illl
nyl5 TGAAGCAATC TGCGCAGCAA TGATTGAGGC GTGGGGATAT CCTGAACTAC
,," 11111 II II'",,~ III '~''',,,,, II .....
,,,,, II ,,,,''"IIII 1
ts CGAGGCAATT TGTGCAGCTA TGATTGAAGC ATGGGGGTAC CCTGAACTGC
'i"i II II IIIII'"
i llt ~ II''"IIII
iiii '"III
iii ~ "IIII: {
ii
n13 CGAAGCTATA TGTGCAGCAA TGATTGAAGC CTGGGGGCAT ACTGAACTCC

401 450
florida TCC~GAGAT T A G ~ G T T C TATCTGTGGT TAC~GAGAG AGATGAGCTG

~ex TCCAAGAGAT TAGAAAGTTC TATCTATGGT TACTTGAGAG AGATGAGCTG

n17 TTCAAGAGAT TAGAAAGTTC TATCTGTGGT TACTTGAGAG AGATGAGCTG

nyl5 TTCAAGAAAT TAGGAAGTTT TACCTGTGGC TACTCGAAAG AGATGAGCTA

I ~IIII ,," II~''"III,,,, II IIIIIII I I~ III",, IIII,,,,
....
ts TCCAAGAGAT TAGGAAGTTT TATCTGTGGC TGCTTGAAAG GGATGAGCTG
I III II IIIII II 11 IIIII I I I II 11 1
n13 TTACAGAAAT AAGGAAATTC TACTTGTGGC TCATGGGTAAAGAAGAATTT

451 500
florida AGAGAGA~2G CAGCT~d~TGG AGGAGCCCCA TACATAGCAG AGTCGGCACT

mex AAAGAGATTG CAGCTAATGG AGGAGCCCCA TACATAGCAG AGTCGGCACT

nl7 AGAGAGA~G CAGCT~TGG AGGAGCCCCA TACATAG~G AGTCGGCAC~

nyl5 AGGGAGAATG CTGCTAGCGG AGGAGCCCCA TATATAGCAG AGTCAGCACT

II I I1'1', ~I IIIIIII1,1 II lllltl:lllllll,l I~ I11 I'~tllll ~lllllll~lllll
ts AGAGAGATTG CAGCTAGTGG AGGAGCCCCA TACATAGCAG AGTCAGCACT

n13 AAGGAATTAG CTTTGAATGG AAAAGCACCA TACATAGCAG AAACAGCCCT

501 550
florida CAAAACTCTT TACACAAACA AGAAAACAAA GATTGAAGAG TTGGCGAAGT

mex CAAAACTCTT T A C A C ~ C A N G ~ C ~ GATTGAGGAG ~ G G C G ~ G T

n17 C ~ C T T TACAC~CA A G ~ G C ~ J ~ G GATTG~GAG ~GGCGgJkGT

nyl5 TAAAACTCTT TACACAAACA AGAGAGCAAA GATTGAAGAA TTGGCAAAAT

ts T~AAACTCTT TATACTAATA AGAAAACAAG GATTGAAGAG TTAGCGAAAT

n13 TCGCAAGCTC TATACGGACA AAGATGCCAA AATGGAGGAA ATGCAAGAGT

551 600
florida AT~G~GT GCTTGATTTT GACTATGAGG TAGGATGCGG AGAGTCTGTG

mex ATCTTGAAGT GCTCGATTTT GACTATGGGG TAGGATGCGG AGAGTCTGTG

n17 A T O ~ 2 G ~ G T GCTCGA~I~2T GACTATG~G TAGGATGCGG AGAGT~G~X~

nyl5 ATCTC~GT GC~CGA~I~ ~ATGAGG TAGGATGCGG A G ~ T C T G ~ 3

~g .... 'III ' ~ " I II llll g~ I II~'''"~I II ~'' Illl
ts ATC~I~G~GT G ~ d ~ C ~ C GA~ACG~G TAC~ATGCC~ AG~T~I~3TG

n13 ACCTGAAACA GCTTGAATTC GATTCTGATG ATGAGGTGTA TGAATCCGTG

601 650
florida CATCTAC~T CAGGATCCGG ACACCCACCA CCACCAGTAG TGGATGCTGG

mex CATCTACAAT CAGGATCCGG ACACCCACCA CCACCAGTAG TGGATGCTGG

ii ii i Ii iii~i
n17 CATCTGCAAT CAGGAATCGG ACACCCACCA CCACCAGTAG TGGATGCTGG

nyl5 CACCTACAGT CAGGAGCTGG ACAACCACCA CCACCAGTAG TGGACACTGG

ts CACCTACA_AT C A G G A C C T G G A C A G C C A C A G C C A C C A A T A G T A G A T G C T G G

n13 TCAACACAAT CCAGCAAGAAAGA AGAAG AGAAAGACGC

651 700
florida TGTC4~ACACT GGG~Q~GACA A G / ~ G A C ~ GAGCAGCAGA G G ~ G G A T C
ill ii 11 IIit IIIII I Ill I III 111tl
,~,II~II III II,,,,,II, ,,, II II, il
mex TGTGGACACT GGGAAGGACA AGAAAGACAA GAGTNGTAGA GGAAAGGATC
iii ii iiii illl, iiiii
n17 TGTGGACACT GAGAAGGACA AGAAAGACAA GAGTAGCAAA GGAAAGGGCC

nyl5 TGTGGACACT GGGAAGGACA AGAAAGATAA AGGCAGTAAA GGAAAGGACC

iii i1 t11t
,,,III 11111,,,,I 1 I.....
llll
I ,,I I I IiIi 1 III I 1 II11 I I 1 1
ts TGTGGAATCT GGGAAGGACA AGAAAGAGAA AAGCAACAAA GGAAAGGACC
It
n13 TGGGGCCGAT GAGAGAGAGA AGGA . . . . . . . . . . . . CAAAGGCAAAGGCC

Fig. 2 (continued)
 Molecular detection of BCMV and BCMNV 1969

701 750
florida CTGAAAGTAA AGAAGAGACA AGAAACAACA GCCGTGGAAC AGAGAATCCA

mex CTGAAAATAAAGAAGAGACA AGAAATAACA GCCGTGGAAC AGAGAATCCA

i%17 CTGAAAATAAAGAAGAGACA AGCAATAGCA GCCGTGGAAC AGAGAACCCA

nyl5 CTGAAAGCAG GGAAGGGATA AGAACTAACA GCCGCGGAAC TGAGAGTTCA

ts AAGAAAGTAG GGAAGGGGCA GGAAACAACA ACCGTGGTGC AGGGAATTCG

II
1113 CAG ...............................................

751 800
florida ACAATGAGAG ACAAGGATGT GAATGCGGGT TCGAGAGGAA AAGTGGTTCC
IIII'''"I,,,,, I '''''llllllll ,,,,"":illll "Ill,, I'",,, lllll''"l,,,,
mex ACAATGAGAG ATAAGGATGT GAATGCGGGT TCGAGGGGAA AAGTGGTTCC

n17 ACAATGAGAG ATAAGGATGT GAATGCGGGT TCAAAAGGAA AAGTGGTTCC

nyl5 ACAATGAGGG ACAAGGATGT GAATGCTGGT TCCAAAGGAA AAGTTGTTCC

III'"',,,, I II''"IIII,,,, ,,,'"III I",, IIIIII",, I I II""II,,,,
ts GCAATGAGAG ACAAGGATGT GAATGCAGGT TCCAAAGGGA AGGTTGTTCC
I I ,," II II II II II III",, I I II ,," II
n13 ....... C G G A T A A A G A C G T T G G A G C T G G C T C A A A A G G A A A A G T A G T G C C

801 850
florida CCGGCTTCAA AGGATCACAA AGAAAATGAA CTTACCTATG GTGAAGGGGA
llll'~ll'l,,,,, I I I ' ' ' ' ' I I , , , , , I'',, I I I I I I I , ' ' I I I I ~, t , l l l l l l i i i ,
mex CCGGCTTCAA AGGATCACAA AGAGAATGAA CTTACCTATG GTGAAGGGGA
llll"'"l,,,,, II''''"II,,,,,, I ' ' " I I I I I , , , , ,,'~llll ,,,'" IIIIII,,,I111
n17 CCGGCTTCAA AGGATCACAA AGAGAATGAA CTTACCCATG GTGAAGGGGA

nyl5 TCGTCTTCAA AGGATCACAA AAAGAATGAA TTTACCCATG GTGAAAGGAA

ts TCGGCTTCAA AAGATCACAAAAAGGATGAA TTTGCCCATG GTGAAAGGGA

n13 AAGATTGCAG AAAATCACCA AAAAGATGAA TTTGCCTATG GTTGGCGGTA

851 900
florida GTGTGATTCT GAACTTAGAC CATCTGTTAG ATTACAAGCC AGAACAAACT
II,,,, II l'''''"il,,,n,,, ,,,,,'''"lllll ,,"III''"',,,,, IIII''''",,,,,,
Fig. 2. Alignment of nucleotide
mex ATGTGATCCT GAACTTAGAC CATCTGTTAG ATTACAAGCC AGAACAAACT sequences of RT-PCR products
n17 GTGTGATCCT GAACTTAGAC CATCTGTTAG ATTACAAGCC AGAACAAACT generated from TS, NY15,
nyl5 ATGTGATCTT GAATTTAGAT CATCTGTTGG ATTACAAGCC AGAACAAACT NLT, Florida, and Mexican iso-
ts ATGTGATCTT AAATTTAGAT CATCTATTGG ATTACAAGCC AGAACAAACT lates of BCMV, and the NL3
ni3 GGATGATTCT A~CTTGGAC CACCTAATTG AGTACAAACC GCAGCAGACG isolate of BCMNV. Binding
florida
901 950
GATCTTTTCA ACACAAGAGC AACAAAGATG CAGTTTGAAA TGTGGTACAA
sites for upstream primers
mex GATCTTTTCA ACACAAGAGC AACAAAGATG CAGTTTGAAA TGTGGTACAA
Ubcmv, Unyl5, and Unl3 in
n17 GATCTTTTCA ACACAAGAGC AACAAAGATG CAGTTTGAAATGTGGTACAA
II~llll subsequent RT-PCR reactions
nyl5 GACCTCTTTA ACACAAGAGC AACAAAGATG CAGTTTGAGA TG~TACAA
are underlined (e.g., nucleotides
ts GATCTTTTTA ACACAAGAGC AACAAAGATG CAGTTTGAAA TGTGGTACAA
106 to 130, NL7). Note restric-
n13 GACTTGTACA ACACAAGAGC TACCAAGGCA CAATTTGAAA GATGGTACGA
tion sites inserted into primers
951
(Table2), accounting for se-
florida TGCTGT
I111~1
quence differences from those
mex TGCTGT above. Sequences are recorded
I~tll
nl7 TGCTGT in GenBank Nucteotide Data-
nyl5 TGCTGT base, accession numbers
ts TGCTGT US5315, US5316, US5317,
n13 AGCAGT US5318, US5319 and US5320

using nucleic acid extracts from infected plant tissues. Primer pair
D b c m v / U b c m v generated a predicted 1456bp product from all 17 B C M V
isolates. N o detectable 1456 bp product was amplified from tissues infected with
B C M N V isolates, in parallel tests (Fig. 4A). Similarly, primer pair Dnl3/Unl3
amplified a predicated 922 bp product exclusively from all B C M N V isolates
(Fig. 4B). Primer pair D t s / U n y l 5 amplified a 1164 bp D N A product from all four
1970 L. Xu and R. O. Hampton

1 5o
florida RWDNSLM~ MSVYYSCHKV GWSDEDIQER LVFFANGDDI ~LSIQEVDLW

mex RWDNSLMVV MSVYYSCHKV GWSDDDIQER LVFFANGDDI ILSIQEVDLW

llllll III l l l l l l l l l l IIII lllll l l l l l l l l l l l l l l l l i l l l
IIIIII III I111111111 IIII IIIII IIIIIIIIII IIIIIIIIII
n17 RWDNSFM~ MSVYYSCHKV GWSDEDIQER LVFFANGDD[ ILSIQEVDLW
i"lll~
t ,,,'" I I I I '''~''~+++,, ll'""Itl,,,,~ +,+~111 '~',,, lltlll''",+,,
nyl5 RWDNSLMVV MSVY¥SCHKVGWSDEDIQER LVFFANGDDI ILSIQEVDLW
,++'fill ,,+'" +llt ++++''++,,,, ll''"'ll+,,,,, ,,,,+If'",,, lllll+ ,,,+"
ts RVVDNSFMVV MSVY¥SCHKV GWSDEDXQER LVFFANGDDI ILSIQETDLW

n13 RVVDNSLMVV ISMY¥SCIKE GWTYDDIQER LVFFANGDDI ILAVQKEDVW

51 i00
florida VLDTFAASFK ELGLNYNFDE RTRKREDLWF MSHCAIQVDG I ¥ I P K L E P E R

mex VLDTFAASFK ELGLNYNFDE RTRKREDLWF MSHCAIQVDG I¥IPKLEPER

n17 VIAY2FAASFK E L G L N Y N F D E RTRKREDLWP MSHCAIQVDG IYIPKLEPER

nyl5 VLDTFAASFK ELGLNYNFDE RTI~REDLWF MSHCAIQVDG V~IPKLEPER

lltlll tl :lll""ll,,,, II , , " l l l l l ,,"llll ,,,'" lll+l''",,,,
ts VLDTFATSFR ELGLNYNFDE RTKKREDLWF MSHCAIEVDG IYIPKLEPER
t tl II~I",, t I I ,,"l l'l, ,,"l I I Ill[lit ,,"
nl3 LYNTLSNSFK ELGLNYDFXE QTTKREXLWF MSHQAMLIDD [YIPKLEQER

io1 150
florida VVSILEWDRS KEMMHRTEAI CAAMIEAWGY PELLQEIRKF YLWLLERDEL
ll+~l'''"++,,+ lIl+,,+,,+~ l,,,~,~]l] ,,,~+'+ll ''+','++ ~ l ~ l ''++'',,
mex WSILEWDRS KEM~RTEAI CAAMIEAWGY PELLQEIRKF YLWLLERDEL

n17 VVSILEWDRS KF~HRPEAI CAAMIEAWGY PELLQEIRKF YLWLLERDEL

nllnlnnlll lllltl ill nlllll+l++ Itlll llll l l l l l l l * l l
nyl5 VVSILEWDRS KEMMHRTEAI CAAMIEAWGY PELLQEIP~F YLWLLERDEL

ts %WSILEWDRR KEMMHRTEAI CAAMIEAWGy PELLQEIRKF YLWLLERDEL

ll~:+,,+
.... tt l++,+l~ l+'+'ltll,,,, lt~ l ''+',,,+ lilt l
n13 IVSILEWDRS KELMI]RTEAI C A A M I E A W G H TELLTEIRKF YLWLMGKEEF

151 200
florida REIAANGGAP YIAESALKTL YTNKKTKIEE LAKYLEVLDF DYEVGCGESV
llll,,,,,' llll,,,,tl t''"llll+,,+, , , " t r i l l ''+,,, II Ill'"',,,,
mex KEIAANGGAP YIAESALKTL YTNKKTKIEE LAKYLEVLDF DYGVGCGESV
till .....
ll+[l lit'If'Ill
fill I''"
fill III lt[lll'"'
illf It III''"
llll
n17 REIA~GGAP ¥IAESALKTL YTNKNARIEE LAKYLEVLDF DYEVGCGESV
~I ....
,++, tl~,,,+,ll l''"l,,,, Ill ++++ttt~''+',+,, l~ll '+''',,,,,
nyl5 RENAASGGAP YIAESALKTL YTNKRAK~EE LAKYLEVLDF NYEVGCGESV
l~ ~t,,,,+
..... l~l,,,,,ll I ''~,,, ' Ill +,"fill",, I t~lll ~'',,,
ts REIAASGGAP YIAESALKTL YTNKKTRIEE LAKYLEVLNF DYEVC-CGESX
I [ I II I[II ,," I II I It ,," ,' ,' I ,,"
n13 KELALNGKAP ¥1AETALRKL YTDKDAKMEE MQEYLKQLEF DSDDEVYESV

201 250
florida HLQSGSGHPP ppWDAGVDT GKDKKDKSSR GKDPNSKEET RNNSRGTENP
lllll ''+'~+++'+ ~tl,,~++~ :,,,,+l~l~ ,+,'"ll ++++'+++llll"+++[+,+++
mex HLQSGSGHPP ppVVDAGVDT GKDKKDKSSR GKDPENKEET RNNSRGTENP

n17 HLQSGIGHPP PPWDAGVDT EKDKKDKSSK GKGPENKEET SNSSRGTENP

till I ,," l l l ~ l ,,"It l'"'~,,,, It ,," II l I It'",,,
nyl5 HLQSGAGQPP PPWDTGVDT GKDKKDKGSK GKDPESREG~ RTNSRGTESS

ts HLQSGPGQPQ PPIVDAGVES GKDKKEKSNK GKDQESREGA GNNNRGAGNS

n13 STQSSKK. . . . . . . EEEKDA G_ADEREK.DK GKGPA . . . . . . . . . . . . . . .

251 300
florida TMRDKDVNAG SRGKVVPRLQ RITKKMNLPMVKGSVILNLD HLLDYKPEQT
:Ill[ ,,,,,
..... Ill',,,,,
.... II I l'lll' lll[l lilll II llllllll }llll'"l[,,,,
mex TMRDKDVNAG SRGKVVPRLQ RITKRMNLPM VKGNVILNLD HLLDYKPEQT
llllll'+llll,] 1 llllltllll,l l l 1 1 1 l l l l l l l l l 'liil lllll',ll, IIllllll'1111'
n17 TMRDKDVNAG SKGKVVPRLQ RITKRMNLPM VKGSVILNLD HLLDYKPEQT

nyl5 TMRDKDVNAG SKGKVVPRLQ RITKRMNLPM VKGNVILNLD HLLDYKPEQT

Fig. 3. Alignment of putative
ts
}l++,,,++
...... tll,,+,,'lt
AMIIDKDVNAG S K G K V V P R L Q
,+,,+t++l
KITKRMNLPMVKGNVILNLD
,,,+"Ill ''++,'+, + I t : + ,.,.,.,.,.
HLLDYKPEQT
amino acid sequences of RT-
n13
l''',,,
...DKDVGAG
,'] l++'''"ll,,,,,
SKGKVVPRLQ
++'+,,, I l l +'++ I + )'"+,,,, +] ,,,''' ,,''
KITIZ/~NLPM V G G R M I L N L D H L I E Y K P Q Q T
PCR products generated from
301 318
TS, NY15, NL7, Florida and
florida DLFNTRATKM QFEMWYNA Mexican isolates of BCMV and
lllll''"l,,,, llll I. .l.l.,
~ex DLFNTRATI<M Q F E M W Y N A the NL3 isolate of BCMNV.
n17 DLFNTI~TKM QFEMWyNA The N-terminal regions of the
nyl5 DLF~TRATKM QFEMWYNA coat protein are underlined
I~III '"l'',lll, [ l l l l l l l
ts DLFNTRATKM QFEMWYNA (Shukla et al+ [14]), beginning at
II Ill ,," III II ,
hi3 DLYNTRATKA QFERWYEA amino acid 203
 Molecular detection of BCMV and BCMNV 1971

Fig. 4. Tests of RT-PCR amplification of 22 BCMV and BCMNV isolates with primer pairs
Dbcmv/Ubcmv (A), Dnl3/Unl3 (B), and Dts/Uny15 (C), showing predicted sizes of PCR
products, respectively. Non-specific smears irregularly appeared as artifacts in nucleic acid
extracts from both infected and healthy-plant controls (not shown). No PCR products
illustrated above occurred as either heterologous or healthy-plant reactions

isolates of BCMV-PG-V (NY15, NY15(prov), NY15(dean) and NY15(zau) but

not from other BCMV pathogroups (Fig. 4C). None of the primer pairs
generated specific PCR products from nucleic-acid extracts of non-infected
plant tissues (data not shown). Non-specific smears, visible below PCR products
1972 L. Xu and R. O. Hampton

may have comprised primer components, or partial or fragmentary amplified

sequences, and ranged in size from 250 to 550 bp equivalents.

Sensitivity of RT-PCR and detection of mixed infection

When a dilution series of purified viral RNA of isolates TS, NY15 and NL3 was
used for amplification by primer pairs Dbemv/Ubcmv, Dts/Unyl5 and
Dnl3/Un]3 respectively, the minimal amounts of viral RNA required for detec-
tion were 10 pg for primer pairs Dbcmv/Ubcmv and Dnl3/Unl3 and 100 pg for
primer pair Dts/Unyl5 (data not shown). Experimental detection of mixed viral
infections was performed by RT-PCR using primer pairs of Dbcmv/Ubcmv and
Dnl3/Unt3 either individually or mixed together. Each primer pair, when used
individually, yielded a band of homologous viral cDNA from intentional
mixtures of BCMV and BCMNV RNA. However, when the primer pairs were
used together, only BCMNV cDNA was generated (i.e., no RT-PCR product of
BCMV was produced; data not shown).

Restriction endonuclease analysis

Results of restriction endonuclease digestion of BCMNV and BCMV RT-PCR
products are illustrated in Figs. 5 and 6 and summarized in Tables 3 and 4. When
applied to BCMNV PCR products, restriction enzye XbaI differentiated the two
BCMNV pathogroups, PG-III and PG-VI (Table 3), by distinct cut sites; and
thus distinct digestion products (Fig. 5). None of the four restriction enzymes
generated profiles precisely differentiating the five BCMV pathogroups; how-
ever, some were potentially useful (Table 4). For example, EcoRI detected
common cut sites in RT-PCR products from all isolates of BCMV-PG-I and
three isolates (NL6, ID123 and NW63-108) of PG-IV, but not from PG-II, V, or
VII isolates (Table 4; Fig. 6A). Thus, digestion products of EcoRI differentiated
BCMV PG-I from PG-II, V and VII. Similarly BamHI cut sites differentiated
isolates of BCMV PG-VII from those of PG-I, II, and V (Table 4; Fig. 6D).

Fig. 5. Results of restriction endonucleaseXbaI digestion of RT-

PCR products amplified from BCMNV isolates (left five lanes)
and two BCMV isolates. Note differentiation in digestion-
product sizes between BCMNV pathogroups PG-III and PG-VI
Molecular detection of BCMV and BCMNV 1973

Fig. 6. Results of restriction

endonuclease digestion of
RT-PCR products amplified
from BCMV isolates by
EcoRI (A), XbaI (B), EcoRV
(C), and BamHI (D). Note the
distinction of digests from
RT-PCR products of PG-I
isolates and three isolates of
PG-IV, vs. those of PG-II,
PG-V, and PG-VII
1974 L. X u a n d R. O. H a m p t o n

T a b l e 3. D i g e s t i o n o f B C M N V RT-PCR products by restriction endonucleases

XbaI, EcoRI, BamHI, a n d EcoRV a

Strains Pathogroup XbaI EcoRI BamHI EcoRV

NL8 (Bash) III - + m

NL8 (Drij) III - +

NL3 VI + + m

NL5 VI + + m

TN1 VI + + w

+ cut; - not cut

T a b l e 4. D i g e s t i o n o f B C M V , R T - P C R products by restriction endonucleases

XbaI, EcoRI, BamHI a n d E c o R V ~

Isolates Pathogroup XbaI EcoRI BamHI EcoR V

Ts I + + - -
Type I + + - -
Iran I + + - -
NL1 I + + - -
PV25 II + - - -
NL7 II . . . .
Florida IV - - + +
Western IV . . . .
NL6 IV - + - +
ID123 IV + + - -
NW63-108 IV + + - -
NY15 V - - - +
N Y 15 ( P r o v ) V - - - +
NY15 (Dean) V - - - +
NY15 (Zau) V - - - +
Mex VII - - + +
NL4 VII - - + +

a+ cut; - not cut

Discussion

Although only one fifth of the BCMV or BCMNV genomic sequences were
known, applications of RT-PCR and restriction endonuclease methodologies
demonstrated the potential to differentiate closely related viral variants, without
complete sequence information. Even minute genomic differences were precisely
 Molecular detection of BCMV and BCMNV 1975

P(DnL3/UN3) i . _ ~
...... J
Xbal
RE
_• BCMNV-PGV!,,,I,,,J

I BCMNV-PGIII I

RT-PCR oo., + lscMv-PGUV, o6.°wl

CMNVl le(Dbcmv/Ubcmv)~~ R__E
~PCR +
V, ]
~'~----[BCMV-PGVlI,
PIDts/Un¥15)

PlDts/Uny151 ~._[,BCMV.PGV i I - ,BCMV-PG , ,,,,',V,,v i,n6,w,nw '

Xbal I--~'tBCMV-PGI, 112s,lVi,nw t

IEcoRV ~ BCMV-PGV,VIt,lVf,n6,1

Fig. 7. Schematicdiagram for detection and differentiationof BCMV, BCMNV, and their
pathogroups by RT-PCR and restriction endonuclease analysis. P Primer; R E restriction
endonucleasedigestion; + cut by endonuclease;- not cut by endonuclease.Pg-I, II, IV, V,
and VII pathogroups of BCMV and PG-III and VI pathogroups of BCMNV. Pathogroup
subscripts identifyBCMV isolates: i ID123; n6 NL6; nw NW63-108; f Florida; w Western;
n7 NL7; 25 PV25

detected using primers designed from sequences established from RT-PCR

products. The specific detection of NY-15 isolates by primer pair Dts/Unyl5 is
an example where a single T-end nucleotide differentiated these isolates from all
other isolates tested.
Experiments to detect mixed virus infections with two primer pairs in the
same tube resulted in only one RT-PCR product, suggesting that there is
probably both competition for nucleotides in the reaction mixture and cross-
interference in template and product assemblies.
Putative N-terminal amino acid sequences differed in both length and
diversity between BCMV and BCMNV isolates, but differed much less among
isolates within BCMV (Fig. 2). These results support the concept that BCMNV is
a distinct entity, apart from BCMV [ 11, t2], as also concluded in recent potyviral
taxonomic changes [i3].
Khan et al. [5] noted that the aphid transmission motif DAG was absent in
their isolate of BCMV NY-15. This motif was also missing in the NY15 isolate of
the present study. The absence of DAG in NY- 15 isolates of both studies suggests
either that laboratory isolates of NY-15 have inadvertently lost this motif or that
survival of NY-15 was perhaps not as dependent on this motif as other BCMV
variants (i.e., perhaps greater facility for seed transmission).
1976 L. Xu and R. O. Hampton

In examining the genomic 3'-terminal regions of pathogroup representatives,

it was recognized that pathotype determination probably resided elsewhere in
the potyviral genome ([8]; other work in our laboratory). Thus, 3'-end distinc-
tions among isolates were utilized as temporary substitutes for preferable
pathotype-determinant sequences, not yet known. Molecular detection that is
truly specific for BCMV and BCMNV pathogroups must therefore await
sequencing of these viral genomes and determination of pathotype-specific
sequences. Likewise, more isolates of each pathogroup than the two to five
included in our study should be compared, in developing pathogroup-specific
probes or procedures. Our pathogroup representatives, however, responded to
RT-PCR with surprising uniformity, facilitating both specific and precise results
(Fig. 4). We demonstrate in this report that applications of RT-PCR, combined
with restriction enzyme analysis of specific PCR products, detect and differen-
tiate BCMV and BCMNV, as well as some pathogroups of these viruses.

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Authors' address: Dr. R. O. Hampton, 2170 Bonnie Drive, Payette, Idaho 83661, U.S.A.
Received February 14, 1996