Virus diseases of vegetable crops in southern Bulgaria

Abstract

Virus diseases of vegetable crops (mainly tomato, cucumber, pepper and phaseolus bean) were surveyed
in 1999 in south-eastern Bulgaria. The most widespread viruses were Tomato mosaic virus (ToMV), Cucumber mosaic
virus (CMV) and Pepper mild mottle virus 1.2 (PMMV 1.2) for pepper; ToMV, CMV and Tomato spotted wilt virus
for tomato; CMV for cucumber; Bean common mosaic virus (BCMV) for bean. Using differential cultivars as well as
ELISA and PCR, the viruses were classified into pathotypes, strains and subgroups. The CMV isolates were of
subgroup I, Iγ and II. The BCMV isolates were identified as temperature-dependent necrotic strains. Bean common
mosaic necrosis virus was found in only two samples. PMMV 1.2 was detected in pepper. Clover yellow vein virus in
bean and Cucumber leaf spot virus in cucumber are here reported for the first time in Bulgaria. A cucumovirus,
different from CMV, was detected in bean. Two unidentified viruses (one with capillovirus-like particles from pepper,
and one with 30 nm spherical particles from cucumber) are currently under investigation.

Full text

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Phytopathol. Mediterr. (2003) 42, 3–8
Introduction
Vegetable crops are important in Bulgaria. In
recent years about 12,570 ha have been occupied
by vegetables, mainly tomato (22%), pepper (17%),
onion (11%), cucumber (9%) and phaseolus bean
(4.7%). These crops are generally grown in the
open, with a comparatively small acreage in tun-
nels or greenhouses. Most vegetable crops are lo-
cated in south-eastern Bulgaria, the so-called “Bul-
garian vegetable garden”, where mild climate and
Virus diseases of vegetable crops in southern Bulgaria
DIMITRINA KOSTOVA1, VITTORIA LISA2, ROBERT G. MILNE2, ANNA MARIA VAIRA2,
GIUSEPPINA DELLAVALLE 2 and STEFANOS TSORLIANIS1
1 Institute for Horticulture and Canned Food, Plovdiv, Bulgaria
2 Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy
Summary. Virus diseases of vegetable crops (mainly tomato, cucumber, pepper and phaseolus bean) were surveyed
in 1999 in south-eastern Bulgaria. The most widespread viruses were Tomato mosaic virus (ToMV), Cucumber mo-
saic virus (CMV) and Pepper mild mottle virus 1.2 (PMMV 1.2) for pepper; ToMV, CMV and Tomato spotted wilt virus
for tomato; CMV for cucumber; Bean common mosaic virus (BCMV) for bean. Using differential cultivars as well as
ELISA and PCR, the viruses were classified into pathotypes, strains and subgroups. The CMV isolates were of
subgroup I, Iγ and II. The BCMV isolates were identified as temperature-dependent necrotic strains. Bean common
mosaic necrosis virus was found in only two samples. PMMV 1.2 was detected in pepper. Clover yellow vein virus in
bean and Cucumber leaf spot virus in cucumber are here reported for the first time in Bulgaria. A cucumovirus,
different from CMV, was detected in bean. Two unidentified viruses (one with capillovirus-like particles from pepper,
and one with 30 nm spherical particles from cucumber) are currently under investigation.
Key words: viruses, phaseolus bean, cucumber, pepper, tomato.
Corresponding author: A.M. Vaira
Fax: +39 011 3977285
E-mail: a.vaira@ivv.to.cnr.it
water availability favour production. Unfortunate-
ly this production is often limited by pests and dis-
eases, among which viruses seem to play an im-
portant role. Earlier surveys on viruses of vegeta-
bles in Bulgaria were conducted by Kovachevski
(1965, 1971), Kovachevski et al. (1977) and Kosto-
va and Dimitrov (1995).
The investigation here reported was carried out
in 1999, mainly on viruses in four crops (cucum-
ber, pepper, phaseolus bean and tomato) grown in
south-eastern Bulgaria. The new data will be used
in the national breeding programme aimed at cre-
ating virus-resistant vegetable lines and cultivars.
Some of the results were briefly reported earlier
(Tsorlianis and Kostova, 2000; Tsorlianis et al.,
2000; Kostova et al., 2001).
RESEARCH PAPERS

4Phytopathologia Mediterranea
D. Kostova et al.
Materials and methods
Collection of virus samples
A total of 102 samples from different crops
were collected; of these 18 were from cucumber,
25 from phaseolus bean, 38 from pepper, 17 from
tomato, three from melon and one from eggplant.
For each crop, except melon and eggplant, sam-
ples were collected in at least four locations. Sam-
ples were collected in June 1999 and consisted
of leaves taken from plants with symptoms sug-
gesting virus infection. Leaves were placed in
plastic bags and stored in a cool bag in the field.
Upon arrival in the laboratory at the Institute
for Horticultural and Canned Food in Plovdiv,
samples were divided in two parts: one was des-
iccated over CaCl2, and the other under vacuum.
All desiccated samples were then taken to the
Istituto di Virologia Vegetale (IVV) (formerly Is-
tituto di Fitovirologia Applicata) in Torino, Ita-
ly, where they were analysed under strict phy-
tosanitary control.
Isolation and host-ranges
To prepare inoculum, the dried samples were
ground in ice-cold 50 mM phosphate buffer, pH 7,
containing 5 mM Na-DIECA, 5 mM Na-thioglyco-
late, 1 mM Na-EDTA and about 10 mg ml-1 acti-
vated charcoal. The sap was rubbed on carborun-
dum-dusted leaves of Chenopodium quinoa, Nico-
tiana benthamiana, N. clevelandii and ‘White Bur-
ley’ tobacco for all samples, plus Capsicum annu-
um cv. Quadrato d’Asti for pepper samples, Cucu-
mis sativus cv. Marketer and Cucurbita pepo cv.
Genovese for cucumber, Lycopersicum esculentum
cv. Marmande for tomato and Phaseolus vulgaris
cv. Saxa for bean.
Viruses were isolated from mixed infections by
differential reactions of the host plants or, for some
viruses, by thermal inactivation (10 min at 75°C)
of the more unstable virus.
Capsicum spp. differentials (C. annuum ‘Bru-
insma Wonder’, C. frutescens ‘Tabasco’, C. chi-
nense PI 159236 and C. chacoense PI 260409) were
used to pathotype tobamoviruses (Rast, 1988),
and bean differentials (P. vulgaris ‘Widusa’, ‘Ju-
bila’, ‘Amanda’ and IVT 7233) to differentiate
Bean common mosaic virus (BCMV) pathotypes
(Morales and Bos, 1988) and to confirm the iden-
tification of Bean common mosaic necrosis virus
(BCMNV).
Serological tests
Original desiccated samples of each crop were
tested for several viruses by different ELISA pro-
cedures, as indicated in Table 1. ELISAs were per-
formed according to standard procedures (Converse
and Martin, 1990), with dilution of reagents ac-
cording to manufacturer’s instructions or IVV pro-
tocols. Reactions were considered positive when the
infected/healthy ratio was 3 or more.
Agar gel diffusion (done according to Purcifull
and Batchelor, 1977, for the elongated viruses) was
used for the detection of some viruses in sap from
experimentally infected plants. Antisera to BCM-
NV, Broadbean wilt virus-1 and -2 (BBWV-1, -2),
Clover yellow vein virus (ClYVV) and Tomato bushy
stunt virus, were from the IVV collection; antisera
to Cucumber leaf spot virus (CLSV) was from D.
Gallitelli, Bari, Italy, to Cucumber necrosis virus
from DSMZ, Braunschweig, Germany, to Cucum-
ber soil-borne virus from R. Koenig, Braunschweig,
Germany, and to Sowbane mosaic virus (SoMV)
from C.I. Kado, Riverside, CA, USA.
Electron microscopy
Extracts of original desiccated samples and ex-
perimentally infected indicator plants were tested
by negative staining with uranyl acetate.
RT-PCR analysis of CMV isolates
Approximately 0.2 g leaf tissue from experi-
mentally infected test plants of selected samples
were processed with the RNAWIZ reagent (Ambi-
on, Austin, TX, USA) according to manufacturer’s
instructions, to obtain total RNA. The RNA pellets
were resuspended in 40 µl diethyl pyrocarbonate-
treated water and 1 µl was used as template for
reverse transcription. Complementary DNA syn-
thesis and PCR were performed as described in
Anonymous (1998), using 5’CP and 3’CP primers
(Rizos et al., 1992) and MspI digestion of the am-
plicons. After enzymatic digestion, the DNA was
electrophoresed in 3% NuSieve 3:1 agarose (FMC,
Rockland, ME, USA) and stained with ethidium
bromide.
Results
A total of 102 samples were tested. The viruses
detected, both in the original desiccated material
by ELISA and those recovered from experimental-

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Vol. 42, No. 1, April 2003
Viruses of vegetables in Bulgaria
ly infected indicator plants, are listed in Table 2.
Most data obtained on the original material by
ELISA could be confirmed by infectivity tests, with
the possible exception of Alfalfa mosaic virus
(AMV), for which no good correlation between ELI-
SA and infectivity was found. No appreciable dif-
ference was found between samples desiccated
under vacuum and those treated with CaCl2. A
number of samples were negative by ELISA for the
viruses tested, but infectivity tests showed them
to be infected with other viruses such as BBWV-1,
ClYVV, or two unidentified viruses. Some viruses,
especially potyviruses such as Potato virus Y (PVY),
Watermelon mosaic virus (WMV) in one case, or an
unidentified potyvirus detected in eggplant by
ELISA, could not be isolated on the indicators. As
expected, Tomato spotted wilt virus (TSWV) infec-
tivity did not survive in the desiccated leaves. About
Table 1. Antisera or antibodies used to detect viruses by ELISA in the original desiccated samples.
Crop Virus Type of ELISA and reagents
Cucumber Cucumber mosaic (CMV)TAS, coating: sera to subgroup I + IIa; Mabs: 2+85b; Mabs subgroup I/IIc
and melon Cucurbit aphid-borne yellowing DAS, kit supplied by H. Lecoq, Montfavet, France
Moroccan watermelon mosaic DAS, serum supplied by Dr S. Winter, Brauschweig, Germany,
purified and conjugated at IVV
Papaya ringspot DASa
Tobacco necrosis (TNV) DASd
Squash mosaic DAS, Loewe Biochemica GmbH, Sauerlach, Germany
Watermelon mosaic (WMV) DASa
Zucchini yellow fleck DASa
Zucchini yellow mosaic DASa
Watermelon silver mottle TAS, coatinga, Mab 1B4e
Bean Alfalfa mosaic (AMV) DAS, Loewe Biochemica GmbH
Bean common mosaic (BCMV) DASa
Bean yellow mosaic DAS, Loewe Biochemica GmbH
CMV TAS, coating: sera to subgroup I + IIa; Mabs: 2+85b; Mabs subgroup I/IIc
TNV DASd
Cucumovirus Ch39 DASa
Pepper AMV DAS, Loewe Biochemica GmbH
CMV TAS, coating: sera to subgroup I + IIa; Mabs: 2+85b; Mabs subgroup I/IIc
Pepper mild mottle (PMMV) 1,2,3 DASa f
Potato virus Y (PVY) DASa
Tomato mosaic (ToMV) DASa g
TNV DASd
Tomato spotted wilt (TSWV) TAS, coating: sera a; Mab NUV2e
Tomato As for pepper, plus
Tomato infectious chlorosis DASa h
Parietaria mottle-T ACPa i
aCollection of the Istituto di Virologia Vegetale, CNR, Torino, Italy.
bGrassi et al., 1995.
cAGDIA kit able to differentiate CMV subgroups I and II.
dRoggero and Lisa, 1995.
eRoggero et al., 1998.
fSerum able to detect all pepper tobamoviruses except P 1.
gSerum able to detect pepper tobamovirus P 0.
hDellavalle et al., 1995.
iLisa et al., 1998.

6Phytopathologia Mediterranea
D. Kostova et al.
19% of the samples were infected by two or more
viruses, while 23% of samples were negative both
in ELISA and in the infectivity tests.
Of the 22 CMV isolates detected by ELISA and
the infectivity tests (Table 2), seven were further
characterised by RT-PCR. Of these, five were iden-
tified as subgroup I and two as subgroup Iγ (Anon-
ymous, 1998). In four bean samples, a cucumovi-
rus similar to the undescribed “cucumovirus Ch39”,
found in 1990 in bean in northern China, was de-
tected by ELISA. This virus was related to, but
different from CMV and from a strain of Peanut
stunt virus and Tomato aspermy virus (P. Roggero,
G. Dellavalle, V. Lisa and F. Morales, unpublished
data). In two of these isolates tested by RT-PCR,
the 870 bp amplicon expected for CMV samples was
not obtained.
Tobamoviruses infecting pepper and tomato
were identified as Pepper mild mottle virus 1.2
(PMMV 1.2) or Tomato mosaic virus (ToMV). BCMV
was the commonest potyvirus detected in bean.
Heat inactivation of CMV or WMV in doubly-
infected indicators permitted the isolation of CLSV
and of an unidentified virus from cucumber with
spherical 30 nm particles, currently under study
at IVV.
A virus with a flexuous capillovirus-like struc-
ture, not yet identified, was found in pepper. In
experimental conditions this virus caused lethal
necrosis on every Capsicum species or C. annuum
cultivar tested. This virus is also currently under
study at IVV.
Discussion
The viruses detected on vegetables in Bulgaria
in this investigation are generally common and
widespread in temperate regions. Some of them,
such as BCMV in bean, or ToMV in pepper and
tomato, were also found to be widespread in the
same crops in Bulgaria by Kovachevski (1965) and
Kowachevski et al. (1977), indicating that some of
the virus problems have not changed substantial-
ly over the last 30 years.
Considering the different crops, the common-
est virus in phaseolus beans in our samples was
Table 2. Viruses detected in 1999 in the desiccated field samples by ELISA and recovered on experimentally infected
plants. Several samples were infected with two viruses.
Desiccated samples (detection by ELISA) Isolation on test plants
Crop No.aVirusbNo.aVirus b
Cucumber 18 CMV c (13), WMV (3), negative (4) 18 CMV (12), CLSV (3), WMV (1), SoMV (1), unidentified
30 nm virus (2), negative (3)
Bean 25 BCMV (11), CMV (7), 25 AMV (2), BCMV (13), BCMNV (2), CMV (6), ClYVV (1),
cucumovirus Ch39 (4), negative (8) cucumovirus Ch39 (4), negative (3)
Pepper 38 AMV (7), CMV (5), tobamoviruses 38 AMV (1), BBWV1 (3), CMV (3), PMMV 1.2 (4), ToMV (15),
(15), TSWV (4), negative (9) unidentified capillo-like virus (1), negative (13)
Tomato 17 CMV (1), PVY (2), ToMV (3), 13 CMV (1), ToMV (4), unidentified carla-like virus (2),
TSWV (7) negative (6)
Melon 03CMV (1) 02Negative (2)
Eggplant 01AMV, unidentified potyvirus 01AMV
aNo. of samples tested.
bIn brackets No. of samples found infected or negative.
cCMV, Cucumber mosaic virus; WMV, Watermelon mosaic virus; CLSV, Cucumber leaf spot virus; SoMV, Sowbane mosaic virus;
BCMV, Bean common mosaic virus; AMV, Alfalfa mosaic virus; BCMNV, Bean common mosaic necrosis virus; ClYVV, Clover yellow
vein virus; BBWV1, Broad bean wilt virus 1; TSWV, Tomato spotted wilt virus; PMMV 1.2, Pepper mild mottle virus 1.2; ToMV,
Tomato mosaic virus; PVY, Potato virus Y.

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Vol. 42, No. 1, April 2003
Viruses of vegetables in Bulgaria
BCMV, followed by CMV, BCMNV and AMV. Such
a virus-distribution for bean has already been re-
ported in Bulgaria (Kostova et al., 1995). BCMV
and BCMNV isolates detected in this study were
further studied at IHCF (Tsorlianis and Kostova,
2000). The BCMV isolates were identified as tem-
perature-dependent necrotic strains, but were con-
sidered to be unique on the basis of their pheno-
typic expression on bean differential varieties. The
BCMNV isolates were similar to the former VIa
pathotype of BCMV (Morales and Bos, 1988). Based
on PCR results, CMV bean isolates were assigned
to subgroup I and subgroup Iγ. The first isolate of
CMV from bean to be assigned to subgroup Iγ was
also from Bulgaria (Anonymous, 1998). The find-
ing of more Bulgarian CMV isolates belonging to
this subgroup suggests that this virus may be en-
demic in the area, as already noted by Tsorlianis
et al. (2000). The finding of a cucumovirus similar
to isolate Ch39, found in 1990 in northern China,
is also of interest. In field beans this virus was asso-
ciated with severe disease, and it should be fur-
ther studied. The detection of ClYVV is a new
record for Bulgaria.
CMV appeared to be the most widespread virus
in cucumber. All isolates tested by RT-PCR be-
longed to CMV subgroup I. WMV, found in cucum-
ber and in one zucchini sample (data not shown),
was already reported in Bulgaria by Dikova et al.
(1984). Two viruses new for Bulgaria on cucumber
were identified by this study: CLSV (Kostova et
al., 2001) and SoMV. SoMV can be considered an
occasional infection since it occurs commonly in
weeds. Four original cucurbit samples showing
yellowing were tested for cucurbit criniviruses (Cu-
curbit yellow stunting disorder virus and Lettuce
infectious yellows virus) by RT-PCR with specific
primers and found to be negative (A.M. Vaira, data
not shown).
In pepper and tomato samples, tobamoviruses,
particularly ToMV, and TSWV, were the viruses
most frequently detected. ToMV appeared to be a
serious problem particularly for pepper, being iden-
tified in about 50% of samples. In both these crops
CMV was also detected. ELISA of CMV isolates
revealed both subgroups, I and II. In pepper, AMV,
BBWV-1 and PMMV-1.2 were also identified, at
lower percentages. These viruses have already been
reported in Bulgaria (Kovachevski, 1976; Yanku-
lova and Kaitazova, 1979; Kostova et al., 1995).
The relatively high number of samples nega-
tive both by ELISA done on original material and
by the infectivity tests (23%) can be explained by
considering that sampling was done early in the
season, after an unusually cold spring that had
delayed the transplanting of seedlings to the field.
Particularly in pepper and tomato crops, the plants
were rather young and the symptoms sometimes
uncertain. Furthermore, viruses not sap-transmis-
sible to indicators, or for which no detection kit
was available at IVV, may have escaped detection.
Acknowledgements
This work was done in the framework of the
XIV Protocol of Scientific and Technological Coop-
eration between Italy (Ministry of Foreign Affairs)
and Bulgaria (Ministry of Education), and we grate-
fully acknowledge the support of both Institutions.
We also thank all those who contributed with an-
tisera.
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