Virus Research 71 (2000) 9 – 21

www.elsevier.com/locate/virusres

The ecology of Cucumber mosaic 6irus and sustainable

agriculture
Donato Gallitelli *
Dipartimento di Protezione delle Piante dalle Malattie,
Uni6ersità degli Studi and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, Via Amendola 165 /A,
76126 Bari, Italy

Abstract

An account is given of the ecology of Cucumber mosaic 6irus (CMV) as a pertinent example of how a virus can
affect the sustainability of an important crop. It is now generally accepted that the technologies used in modern
agriculture should ensure that production systems are operated in such a way that the quality of the produce is
maintained year after year without causing degradation of the environment. Recent experiences in countries of the
Mediterranean basin demonstrate that the benefits expected from the introduction of new and highly productive plant
varieties may be quickly eroded by the concomitant introduction of new virus strains which can greatly change the
structure of the resident virus population. Quarantine inspection of plant propagules and genetic engineering are
suggested as powerful tools to help achieve sustainability. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Cucumber mosaic 6irus; Ecology; Control; Sustainability

1. Introduction Plant viruses infect many crops, causing eco-

The extensive use of pesticides to prevent crop
losses due to pathogens presents a threat to the
sustainability of modern agriculture in which
there is a need to avoid damaging the environ-
ment and future food availability. Agricultural
systems are required that use technologies, inputs
and management practices that will ensure high
productivity, while preserving the quality of the
environment (Herdt and Steiner, 1995).
* Corresponding author. Tel.: +39-80-5443071; fax: + 39-
80-5442911.
E-mail address: gallitel@agr.uniba.it (D. Gallitelli).
nomically important diseases second only to those
caused by fungi. However, while the control of
fungal diseases can usually be based on preven-
tion and cure, that of virus diseases is based solely
on preventive measures. These may be very costly
if they are to be effective. For instance, the popu-
lation threshold level of insects as virus vectors is
much lower than for the same insects as direct
pests (Satapathy, 1998). The occurrence of rela-
tively few individual vectors which do not damage
the crop by their feeding may lead to the intro-
duction and subsequent spread of viruses in the
field, thus enhancing the need for chemical
control.

0168-1702/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 7 0 2 ( 0 0 ) 0 0 1 8 4 - 2
10 D. Gallitelli / Virus Research 71 (2000) 9–21
This paper describes the complex ecology of
Cucumber mosaic 6irus (CMV) and provides a
pertinent example of how a virus can undermine
the sustainability of an important crop.
2. Properties of CMV
CMV, the type species of the genus Cucu-
mo6irus in the family Bromo6iridae (Ribicki,
1995), has polyhedral virions which encapsidate
three linear plus-sense single-stranded genomic
RNAs (RNA1, RNA2, RNA3) and the sub-ge-
nomic RNAs 4 and 4A. RNA1 and RNA2 are
encapsidated in distinct particles, whereas RNA3
and RNA4 (Lot and Kaper, 1976) and possibly
RNA3 and RNA4A are encapsidated together.
Hence, CMV is a multi-component virus for
which inoculation of the three types of particles is
required to infect plants.
RNA1 is monocistronic and codes for a single
product of 110 kDa which is often designated 1a
protein and is thought to have both methyltrans-
ferase and RNA helicase activity. RNA2 encodes
a large product of 98 kDa, designated 2a protein,
which contains the conserved amino acid se-
quence GDD of many viral polymerases. RNA2
also encodes the 2b protein which is translated by
sub-genomic RNA4A. This protein may play a
role in virus transport within plants and acts as an
important determinant of virulence (Ding et al.,
1995, 1996; Brigneti et al., 1998; Li et al., 1999).
RNA3 is also bicistronic encoding the 3a protein
in the 5% proximal half of the molecule and con-
taining the open reading frame (ORF) for the CP
in the 3% proximal half. The CP cistron is trans-
lated via the subgenomic RNA4. The 3a protein,
also designated MP, is involved in virus
movement.
A fifth RNA, denoted RNA5 is also present in
some CMV strains. RNA5 consists of a mixed
population of molecules of c. 300 nucleotides
derived from the imperfectly conserved 3%-termi-
nal region of genomic RNAs 2 and 3 (Blanchard
et al., 1996). CMV RNA5 may play a role in viral
replication (Blanchard et al., 1996, 1997).
In addition to genomic and subgenomic RNAs,
some strains of CMV encapsidate a satellite RNA
(satRNA), which is a small RNA molecule that is
completely dependent on the viral genome for its
replication and spread, but does not supply the
helper virus with any essential function. CMV
satRNAs range from 335 to 405 nucleotides in
size, apparently do not contain any functional
ORF and occur in nature as several variants
which can sometimes attenuate or aggravate dis-
ease symptoms induced by the helper virus (Gar-
cı́a-Arenal and Palukaitis, 1999). This effect is
particularly relevant in tomato where the CMV
infection phenotype may cause no symptoms or
necrosis.
3. Economic relevance
Tomlinson (1987) listed CMV as the virus of
greatest economic importance in celery, cowpea,
cucurbits, lettuce, pepper and tomato. Banana
(Palukaitis et al., 1992; Gafny et al., 1996), pas-
ture legumes (Latham et al., 1999; Jones, 2000),
Kava (Davis et al., 1996) and ornamentals
(Flasinski et al., 1995) are also affected.
CMV appears to be the most important virus of
some annual crops in Argentina, eastern China,
Croatia, France, Egypt, Greece, Israel, Italy,
Japan, Poland, Portugal, Spain, Sweden and in
the north east of US. In other countries, CMV
ranks second or third in importance (Tomlinson,
1987).
In the Mediterranean basin the economic im-
portance of CMV correlates with recurrent out-
breaks in tomato crops grown for canning
(Gallitelli et al., 1995; Varveri and Boutsika, 1999)
and, more recently, in melon (Alonso-Prados et
al., 1997; Luis-Arteaga et al., 1998) and pepper
(Mnari Hattab et al., 1999) with incidences of 30
to nearly 100%. After the tomato necrosis disease
reported in Alsace (France) in 1972, tomato dis-
eases induced by CMV became suddenly impor-
tant in Italy and Spain in 1987 (Gallitelli et al.,
1988; Jordá et al., 1992). There the virus caused
up to 100% crop losses in the main production
areas. To date, a number of reports indicate that
CMV populations are well established in Mediter-
ranean areas where they are frequently found in
mixed infections with other viruses that have ei-
 D. Gallitelli / Virus Research 71 (2000) 9–21
ther a wide (e.g. Alfalfa mosaic 6irus and Tomato
spotted wilt 6irus) or restricted host range (e.g.
potyviruses infecting cucurbits).
4. Ecology
4.1. CMV strains
The existence of many CMV strains that have a
very broad host range suggests that the multi-
component nature of the virus does not constrain
its ecological success or epidemiological compe-
tence (Fulton, 1980). Rather, the consensus is that
random reassortment of intact genomic segments
from multi-component viruses is a parasexual
process generating new variants to counterbalance
losses in fitness due to the to accumulation of
deleterious mutations through successive host pas-
sages. It has been postulated that in a small
asexual population deleterious mutations accumu-
late through an irreversible, ratchet-like mecha-
nism, known as Muller’s ratchet. Genetic
exchange by reassortment and by recombination
may help in rescuing viable virus fitness from a
population that has been ratcheted-down (Rooss-
inck, 1997 and references therein).
Viable reassortants have been prepared in vitro
and used to assign specific functions to individual
segments of the genome (Palukaitis et al., 1992;
Carrère et al., 1999). True recombination (RNA –
RNA recombination) (Fernandez-Cuartero et al.,
1994) and the production of mixed subunit cap-
sids from two different cucumoviruses (Chen et
al., 1995) that may lead to radical recognition
changes in insect-mediated transmission were
demonstrated also for CMV.
Although postulated several years ago, the nat-
ural occurrence of reassortants in the genus Cucu-
mo6irus was demonstrated only recently for Bean
distortion mosaic virus (BDiMV), found in Chile
(White et al., 1995). After a recent survey of CMV
in Spain (Fraile et al., 1997), it was suggested that
reassortment or recombination are rare in natural
populations of this virus and that the reassortant
genomes that have been found do not correspond
to a simple random association of the three ge-
nomic segments. Thus, CMV strains with reassor-
11
tant or recombinant genomes seem not to be
favoured under natural conditions. Although the
nature of this competitive disadvantage is largely
unknown, one hypothesis suggests that, in CMV,
reassortment is selected against aphid transmis-
sion (Fraile et al., 1997; Gallitelli et al., 1997).
Another explanation may rely on the scarcity of
natural mixed infections of different CMV strains
which are required for genetic exchange to occur
in plants.
Strains of CMV can be divided into sub-groups
I and II on the basis of their sequence similarity
and serological relationships. Surveys of naturally
infected crops suggest that sub-group I strains are
more frequent than those of sub-group II and
sometimes they represent more than 80% of all
isolates (Crescenzi et al., 1993a; Fraile et al.,
1997).
Recent sequence data show that a number of
CMV strains within sub-group I and originating
from Asia differ by 7–12% in sequence arrange-
ment from other sub-group I strains. Accordingly,
Palukaitis and Zaitlin (1997) proposed that sub-
group I should be split by placing the ‘Asian
strains’ in sub-goup IB and the others in sub-
group IA. Strains in the same sub-group differ by
only 2–3% of their sequence homology. Phyloge-
netic analyses of CMV using the whole genome or
the ORFs of single genes strongly supports the
sub-division of sub-group I, considering CMV
strains so far sequenced (Roossinck et al., 1999;
Barbarossa, Lanave and Gallitelli, unpublished
results), although the different branching patterns
observed with different ORFs suggest that both
recombination and reassortment may have con-
tributed to the evolutionary history of CMV
(Roossinck, 1999).
4.2. CMV transmission
CMV can be acquired and transmitted by more
than 80 aphid species during very brief and su-
perficial probes of infected and healthy plants.
Such probes are characteristic of aphids and are
used by them in host plant recognition (Pollard,
1973). The probes involve 5–10 s intracellular
punctures of epidermal or mesophyll cells (Martin
et al., 1997 and references therein). The most
12 D. Gallitelli / Virus Research 71 (2000) 9–21
widely accepted hypothesis suggests that CMV
particles are acquired when aphids ingest cell con-
tents during the food selection phase and are then
transmitted to healthy plants during intracellular
egestion or salivation in a subsequent host (Mar-
tin et al., 1997). Thus the possibility that a plant
species on which aphids probe but do not feed
may be a potential new host for the virus can be
regarded as the vulnerable ‘Achilles heel’ in any
attempts to control CMV. McKirdy and Jones
(1999) detected total infection of plants of chick-
pea (Cicer arietinum) surrounding plants of nar-
row-leafed lupin (Lupinus angustifolius) in which
seed-borne CMV served as primary infection foci.
This occurred even though chickpea is not a host
of aphids. Initially, most of the infected chickpea
plants were around primary infection foci, but
within 4 weeks infected plants also occurred at
random away from such foci and served as addi-
tional sources for further spread.
The rate of transmission can be affected by the
virus strain, by the species of aphid (Perry et al.,
1994, 1995) and by the quality of the recipient and
donor plants. This, in turn, is influenced by bio-
chemical changes in metabolism due to virus
infection.
Some CMV strains are not transmissible or are
poorly transmitted by aphids. These differences in
transmissibility are probably not only attributable
to differences in the amino acid composition of
the virus capsid, but also to specific segments of
the genome (Zitter and Gonsalves, 1991) whose
structure may affect protein/protein or RNA/
protein interactions with a dramatic impact on
particle stability. Complete loss or reduced rate of
aphid transmissibility may represent a genetic
‘bottleneck’ for many natural reassortants, a trait
that correlates positively with their rare occur-
rence in nature.
CMV is also borne in the seed of several plant
species. Among cultivated crops, seed transmis-
sion of CMV seems particularly relevant for spe-
cies in the Leguminosae and Cruciferae and, to a
lesser extent, in some cucurbits. There is evidence
that seed transmission in Phaseolus 6ulgaris is
determined by the RNA1, suggesting a relation-
ship between the RNA1-coded product of the
viral replicase complex and seed-transmission.
Lack of host-encoded products of the viral repli-
case complex in embryonic cells could account for
the absence of seed-transmission in a number of
hosts of CMV. There are reports that CMV can
replicate in all reproductive tissues of spinach and
that some seeds probably contain a mixed infec-
tion of two distinct isolates of CMV (Hampton
and Francki, 1992; Yang et al., 1997). The ecolog-
ical significance of mixed infections for seed trans-
mission of CMV strains is still under study.
The rate of seed-transmission in weed species
other than chickpea (Stellaria media) remains
largely unknown, although seeds as well as rhi-
zomes of certain plant species seem crucial for
perpetuating CMV strains in cultivated areas
(Tomlinson and Carter, 1970; Rist and Lorbeer,
1988), or in alternative pulses and pasture legumes
(Latham et al., 1999). Tomlinson and Carter
(1970) assessed seed transmission of CMV in sev-
eral weed species. In S. media, the rate of trans-
mission was 4–29% and the virus was still
infectious after 5 months in infected seeds buried
in the soil. Thus, the virus may overwinter in
seeds of this host and germination of the seeds
leads to infection foci scattered throughout the
field which may serve as sources for subsequent
spread by aphids. It was calculated that if 10% of
seeds germinate, 1% seed transmission will lead to
one infected seedling per square metre (Tomlinson
and Carter, 1970). The seed-borne infection in S.
media is usually asymptomatic, although mosaic
symptoms are caused by some CMV strains. In
addition to S. media, seed transmission of CMV
was demonstrated in Lamium purpureum (4%),
Cerastium holostioides (2%) and Spergula ar6ensis
(2%) (Tomlinson and Carter, 1970).
Although seed transmission has not been
proven or assessed for many weed species that are
susceptible to CMV, their role in providing foci of
infection for subsequent spread was first estab-
lished in cucurbit crops in Wisconsin, USA
(Doolittle and Walker, 1925) and subsequently
(Tomlinson et al., 1970; Quiot et al., 1979; Rist
and Lorbeer, 1988; Crescenzi et al., 1993b). In
some instances, S. media, Senecio 6ulgaris and
Portulaca oleracea were found to be 60–100%
infected (Tomlinson et al., 1970; Quiot et al.,
1979).
 D. Gallitelli / Virus Research 71 (2000) 9–21
Riedle (1995) found that the leaf hairs of cu-
cumber plants contain a high concentration of
CMV particles. This may imply that dispersal of
the virus could occur in the aerosols following
hair breakage, as with contact-transmitted viruses.
However, this possibility has not been
investigated.
5. CMV as a threat to the sustainability of
farming systems: the Italian experience
Italian horticulture is characterized by a great
diversity of annual crops of high commercial
value, among which tomato is particularly impor-
tant. In recent years epdemics of CMV, Tomato
spotted wilt 6irus (TSWV), Alfalfa mosaic 6irus
(AMV) and Potato 6irus Y (PVY) have emerged
as the most serious threats to the economic viabil-
ity of Italian horticultural systems and related
activities (e.g. employment in the canning tomato
industry). Epidemics of CMV are particularly
detrimental to commercial fields of canning
tomato, because new or unusual disease pheno-
types have emerged in addition to the well known
fern leaf/shoestring and tomato necrosis (syn-
onym: tomato lethal necrosis) diseases (Kaper and
Waterworth, 1981). CMV outbreaks have also
been recorded in celery, melon, pepper, tobacco
and zucchini squash.
In tomato, at least two new disease phenotypes
appeared. The one referred to as tomato top
stunting (TTS) was caused by a variant of CMV
satRNA and the other, denoted tomato fruit ne-
crosis (Tfn), was caused by CMV alone.
Top stunting disease is characterized by a
marked shortening of the internodes of the apical
shoots that confers a compact and bushy appear-
ance to the plant. Leaflets are small, with the
lamina rolled upward as the mid-vein curves
downward and twists. Older leaves are of normal
size and exhibit a mild mosaic. Yields are drasti-
cally reduced and the fruits may be unmarketable
as they are few, small and mature unevenly. The
disease phenotype is caused by a CMV strain,
denoted CMV-TTS, supporting a non-necrogenic
satRNA (Grieco et al., 1992). CMV-TTS artifi-
cially deprived of its satRNA induces ordinary
fern-leaf and shoestring symptoms.
13
Fruit necrosis disease is characterized by ex-
tended internal necrosis of the fruits that differs
from the physiological disorder known as ‘inter-
nal browning’ (Blancard, 1988). In most cases, the
plants are vigorous and do not show foliar symp-
toms, except for occasional mild leaf discoloura-
tions. Sometimes, however, plants also show the
fern leaf/shoestring condition typically induced by
ordinary CMV strains. Fruit tissues affected by
necrosis are the mesocarp and particularly those
surrounding the base of the pedicel. Externally,
brown soft blotches correspond to altered tissues.
A CMV strain, denoted CMV-Tfn, was isolated
consistently from tomato plants having symptom-
less foliage and fruit necrosis. CMV-Tfn sup-
ported a 390-ribonucleotide satellite named
Tfn-satRNA (Crescenzi et al., 1993a). This sa-
tRNA was not present in plants showing both
fruit necrosis and fern leaf/shoestring. Its occur-
rence was apparently responsible for the fruit
necrosis condition, but prevented the appearance
of filimorphism on the leaves.
Economically, internal fruit necrosis is even
more damaging than lethal necrosis, which may
cause death of all plants in a field. Unlike lethal
necrosis, where plants show early necrotic symp-
toms, foliage and plant growth seem normal and
farmers are unaware that fruits have internal ne-
crosis and are unmarketable until the fruits ripen,
by which time most of the production costs have
already been incurred. In the absence of Tfn-sa-
tRNA, the fern leaf/shoestring condition was as-
sociated with another strain, denoted CMV-FL
(Crescenzi et al., 1993b).
Similar diseases were also described in Spain
under the name of internal browning and curl-
stunt (Jordá et al., 1992). Infected plants did not
contain Tfn-like satRNA which correlates posi-
tively with the observation that under Spanish
conditions, fruit necrosis was always associated
with fern-leaf/shoestring symptoms.
In Italy, the need to increase the profitability of
crops of canning tomato by reducing costs was
met in the mid-1980s by introducing new tomato
varieties characterized by a short growth cycle
and high yields. Most of these new varieties were
F1 hybrids and particularly vulnerable to viral
epidemics due to their genetic uniformity, as de-
14 D. Gallitelli / Virus Research 71 (2000) 9–21
scribed for other crops (Thresh, 1982). Within a
few years, local tomato landraces and old varieties
were replaced by new genotypes grown in large,
uniform stands. This brought obvious advantages,
for example in the use of machinery and produc-
tion more than doubled. Other cropping practices
routinely applied to improve yields and fruit qual-
ity may have increased the vulnerability of these
new crops which became more attractive to insect
vectors. However, the risk of infection became
even greater when very mild winters recurred for 2
consecutive years, thus leading to enormous in-
creases in overwintering aphid populations and to
CMV epidemics. The most notable outcome of
the interaction between these factors was the sud-
den appearance of necrotic phenotypes that were
co-determined by necrogenic satRNA variants on
the new highly susceptible tomato varieties. After
their first appearance in Alsace in 1972, CMV
strains with necrogenic satRNAs were no longer
recorded, although it is likely that they survived in
small foci where they escaped detection. However,
only recently, it became evident that, in addition
to the necrogenic satRNA strains, new CMV
strains appeared in Italy in the mid-1980s and
that these strains caused most of the economic
damage by inducing internal fruit necrosis.
The following evidence favours this hypothesis:
(i) the nucleotide sequence of CMV-Tfn showed
94 – 100% similarity (Barbarossa, Lanave and Gal-
litelli, unpublished results), both at nucleotide and
amino acid level with the CMV-NT9 strain from
Taiwan (Hsu et al., 1995); (ii) the analysis of
phylogenetic relationships placed CMV-Tfn in the
newly proposed sub-group IB of CMV originating
from Asia; (iii) the RT-PCR RFLP analysis
(Finetti Sialer et al., 1999a) of more that 120
CMV isolates collected in Italy by different labo-
ratories from different plant species proved that
CMV-TTS and CMV-FL were also sub-group IB
strains and confirmed the absence of IB strains
before 1988. Thus there is sufficient circumstantial
evidence that sub-group IB strains of CMV were
introduced to Italy in the mid-1980s and that
since then the structure of CMV biodiversity has
been greatly modified in that the IB strains now
constitute more than 50% of the CMV popula-
tion. Outbreaks of IB sub-group strains recur
annually, affecting also old varieties. Very often
there are mixed infection with other destructive
viruses (e.g. TSWV and necrotic strains of AMV).
Crescenzi et al. (1993a) have shown that during
CMV outbreaks in tomato in the Italian regions
of Basilicata and Campania, weeds played a key
role in determining epidemics of sub-group II
isolates that were maintained in wild plants
throughout the winter. Since CMV isolates of
sub-group II were associated with the lethal ne-
crosis disease in tomato, their absence in summer
correlated positively with the natural disappear-
ance of some weeds at the time and with the
self-destructive nature of lethal necrosis. Addi-
tionally, Daniels and Campbell (1992) reported
that sub-group II strains are more thermosensitive
than sub-group I strains. Therefore, in such con-
dition only the weeds were able to maintain
(probably in their seeds) the inoculum throughout
the summer and until the following autumn.
The situation seemed different with sub-group I
strains for which distinct crops rather than weeds
represented the main sources of inoculum. Crops
rather than weeds seemed also crucial in the epi-
demiology of CMV-satRNA during viral epi-
demics in Italy, because: (i) satRNA sequences
were found only in a few weeds; and (ii) hetero-
geneity among satRNA variants isolated in Italy
between 1988 and 1993 did not differ significantly
from one epidemic episode to the other (Grieco et
al., 1997). This suggests that a crop, rather than a
weed, could have been the initial focus leading to
subsequent epidemic spread of sub-group I CMV
strains and their satRNA.
It is still unclear how and from where these
strains were introduced. One hypothesis is that
they were introduced in the seed of new tomato
varieties. However, this conflicts with the view
that CMV is not or very poorly seed-transmitted
in tomato, although seed transmission in the new
tomato varieties has not been tested. However, IB
strains have been recorded in melon and spinach,
two species in which seed transmission occurs.
Another hypothesis is that the strains were intro-
duced with the seedlings established in small
demonstration plots.
As already mentioned, CMV is readily aphid-
transmissible and epidemics are not restricted to
 D. Gallitelli / Virus Research 71 (2000) 9–21
tomato. The virus spread to cucurbits and pepper
generally co-determines necrotic phenotypes as a
result of synergy with resident viruses of the Po-
ty6iridae (e.g. Zucchini yellow mosaic 6irus, Water-
melon mosaic 6irus and Potato 6irus Y).
Abandonment of traditional tomato-growing
areas, severe crop losses, low product quality and
a huge increase in and often indiscriminate use of
insecticides and herbicides are some of the fea-
tures of what has become an ecological disaster.
6. CMV control through sustainable means
6.1. Diagnosis
Since control of CMV must be largely preven-
tive, it should benefit from the availability of
strain-sensitive, reliable and rapid methods of
virus detection and diagnosis. Both serological
and molecular techniques have been adopted and
applied successfully to diagnosis of CMV to: (i)
ascribe field isolates to one of the two currently
recognized viral sub-groups; (ii) identify CMV
and its satRNA as the cause of specific plant
diseases; (iii) achieve timely virus detection for
quarantine and certification purposes; and (iv)
study virus population dynamics.
Sub-group I and II strains can be differentiated
by several means (reviewed by Gallitelli, 1998)
including serology, molecular hybridization analy-
sis, RT-PCR and by RT-PCR followed by enzy-
matic digestion of the amplified product
(RT-PCR RFLP) (Anonymous, 1998 and refer-
ences therein). Recently, a new RT-PCR RFLP
method proved useful in differentiating CMV
strains in sub-groups IA, IB and II rather than in
sub-groups I and II (Finetti Sialer et al., 1999b).
Thus it may provide a simple way to investigate
the dynamics of CMV populations in nature.
More accurate population analyses and identifica-
tion of distinct satRNA variants can be addressed
by an RNase protection assay (RPA), (Aranda et
al., 1993, 1995; Fraile et al., 1997; Garcia-Arenal
et al., this volume).
Although they are undoubtedly effective and
highly sensitive, RT-PCR and other methods
based on specific sequence identification are labo-
15
rious for routine testing when hundreds of sam-
ples need to be analysed quickly. ELISA and dot
blot hybridization are more suitable for this pur-
pose, especially the latter, as blotted membranes
prepared elsewhere can easily be mailed to a
central laboratory for processing. A dot blot hy-
bridization system using digoxigenin-labelled ri-
boprobes and chemiluminescent detection was
developed for the diagnosis of CMV and five
other viruses in the tests required for the phy-
tosanitary certification of ca.45 million tomato
seedlings in Italy (Saldarelli et al., 1996).
6.2. Control of aphid 6ectors
Insecticide sprays are effective means for con-
trolling aphid populations, but they do not act
fast enough to prevent inoculation of CMV dur-
ing the probing phase on the host plant and in
some cases may stimulate additional aphid move-
ment. However, properly applied treatments can
reduce disease incidence by killing aphids before
they can move to other plants.
Effective aphid control leading to low disease
incidence can be achieved providing that numbers
of alate aphids are low. In temperate regions, mild
winters or heavy spring rainfall followed by rapid
temperature increases greatly favour reproduction
and aphid colonies rapidly become crowded lead-
ing to the emergence of numerous alates. Such
colonies often develop on CMV-infected weeds
and therefore their destruction before transplant-
ing is an important means of preventing virus
outbreaks on annual crops and for achieving an
overall reduction of sources of inoculum and vec-
tors in the field. To be effective, weeds should be
destroyed over large areas, but this is not feasible
in places growing diverse crops and it is an ap-
proach that is strongly opposed by some
environmentalists.
A more sustainable control approach is to pre-
vent aphids reaching plants. This is relatively easy
to achieve in nurseries where the young seedlings
can be grown in screenhouses equipped with dou-
ble doors, insect-proof nets and yellow sticky
traps. Although simple in concept, in practice this
control measure can lead to serious problems by
reducing ventilation, thus leading to a damaging
16 D. Gallitelli / Virus Research 71 (2000) 9–21
increase of temperature in the screenhouse
(Antignus, this volume). Eliminating this problem
by forced ventilation and cooling may be very
expensive.
In the field, mulches usually perform better
than nets because the former cover more than
60% of the soil surface and delay infection, at
least until the extent of the plant canopy cover
significantly decreases the efficiency of the
mulches in repelling aphids. However, older
plants can tolerate virus infection better than
young ones and yield marketable fruits. Some
types of reflective silver mulches are soluble in
water and can be applied by spraying. These have
advantages over the common polyethylene film
mulches as they can cover a larger soil surface, are
biodegradable and can be incorporated into the
soil at the end of the crop season, thus eliminating
the problem of disposal after harvesting the crop
(Summers et al., 1995).
Integrated CMV management strategies should
reduce pesticide inputs through the judicious use
of chemical control and therefore should be en-
couraged. An essential pre-requisite is the
availability of healthy propagules (seeds or
seedlings), although for many crops this is very
difficult to achieve. In the absence of totally virus-
free material, threshold levels of CMV infection
should be established for each crop to minimize
the risk of epidemics (Jones, this volume). Such
information is seldom available. Nutter et al.
(1999) reported that CMV infection greatly de-
creased seed size in narrow-leafed lupin. There-
fore, sieving seed lots to remove small seed may
be beneficial in: (i) significantly reducing the risk
of introducing CMV-infected lupin seeds into
growers’ fields; (ii) increasing the probability of
detecting the virus in subsequent tests on the
seeds; and (iii) speeding up analyses (as only the
small seed fraction will need to be tested) during
quarantine checks (see below). This approach may
be useful with other species in which CMV is also
seed-borne.
In lettuce and tomato, early sowing or late
transplanting and avoiding overlapping cycles of
susceptible crops proved effective in preventing
diseases caused by CMV. Moreover, regular irri-
gation coupled with mulching reduced incidence
of tomato fruit necrosis in southern Italy (re-
viewed by Gallitelli, 1998).
6.3. CMV-resistant 6arieties
An environmentally benign approach to control
plant viruses is to confer resistance by introgres-
sion of a gene or genes from wild species into a
commercial variety. Some CMV-resistant or toler-
ant varieties of spinach, pepper and some cucur-
bits are available. Resistant varieties of tomato
have not yet been obtained, although resistance
genes have been detected in some related species.
Conventional breeding methods are now being
assisted by novel procedures that speed up iden-
tification, characterization, selection and intro-
gression of resistance genes.
Since CMV-resistant varieties are not likely to
be commercially available and widely adopted for
a long time, a different approach based on patho-
gen-derived resistance (Sanford and Johnston,
1985) has been developed recently. Selected genes
can be incorporated into the plant genome by
genetic engineering or a mild strain of CMV can
simply be inoculated into the host plant which
will be cross-protected against virulent strains.
The former strategy has advantages over conven-
tional plant breeding because the genes involved
can be more easily detected as they are carried by
the pathogen itself. Current knowledge on differ-
ent aspects of pathogen-derived resistance and
exhaustive risk/benefit analyses has been reported
in several instances.
Cross-protection has been used successfully in
several instances since it is the most immediate
and convenient response against recurrent CMV
outbreaks of the type occurring in many Mediter-
ranean countries. For CMV, cross-protection can
be obtained using mild strains that may or may
not support a satRNA of the ameliorating pheno-
type. However, the satRNA-free approach should
be restricted to areas where there are no risks of
outbreaks caused by CMV strains supporting ne-
crogenic satRNAs. Otherwise the protective strain
itself will replicate the incoming satRNA with
dramatic consequences (Kaper, 1995). Therefore,
in places where natural necrogenic variants occur,
only mild CMV strains harbouring an ameliorat-
 D. Gallitelli / Virus Research 71 (2000) 9–21
ing satRNA should be used for cross-protection.
A number of these strains have been tested in
small experimental fields in China, Italy, US and
Japan (Wu et al., 1989; Gallitelli et al., 1991;
Montasser et al., 1991; Sayama et al., 1993) and
some of them have been used in commercial fields
in China and Japan. There may be ecological risks
associated with cross-protection using ameliorat-
ing satRNAs, although none was observed in the
several instances in which the technique has been
used.
Although having a number of advantages,
cross-protection shows at least two major disad-
vantages, i.e. (i) plants must be preinoculated
before exposure; and (ii) biological properties of
the protective strain must be routinely assayed for
the timely detection of adverse effects. Transgene-
sis overcomes these problems since the desired
gene is stable when incorporated in the plant
genome and inherited in its progeny. Most of the
CMV-encoded genes (in either functional or non-
functional form), including different satRNA
variants, have been introduced into plant genomes
and are stable in conferring suitable levels of
resistance to virus infection. Although the ten-
dency is to attain a broader spectrum of resistance
against mixed infections, Fuchs et al. (1998)
demonstrated by cost-benefit analysis the eco-
nomic viability of squash cultivars that are resis-
tant to a single virus. Despite the very satisfactory
performance of transgenic plants tested in labora-
tory or small scale field experiments, such plants
have not yet been deployed on a large scale in
agriculture.
One of the most undesirable of the potential
effects is the reassortment or true recombination
between viral sequences transcribed from a trans-
gene and those of an infecting virus. Although
investigated in several instances there is no defini-
tive answer to this fundamental question (Aaziz
and Tepfer, 1999).
6.4. Quarantine inspections and phytosanitary
programmes
One of the main tasks of quarantine is to
prevent the entry of pests and pathogens into
areas where they do not occur or are not widely
17
distributed. Because of the worldwide distribution
and broad host range of CMV, quarantine inspec-
tions and tests may seem inapplicable. Neverthe-
less, quarantine inspections should be directed to
seed germplasm and new accessions of plant spe-
cies in which the virus may be seed-borne. Hamp-
ton et al. (1982) suggested that such new
accessions should be assayed for seed-borne
viruses and that first-generation seed should be
produced in insect-proof screenhouses. If this ap-
proach had been adopted in timely fashion it is
likely to have avoided the accidental introduction
of IB strains of CMV to Europe. Too often the
sanitary status of mother plants is evaluated sim-
ply by visual inspection to detect specific virus
infections and this has been proved to be unreli-
able in several instances. For example, Gillaspie et
al. (1995) found that 70% of cowpea seed lots
from symptomless mother plants of different
genotypes contained one or more seed-borne
viruses, including CMV.
Ornamental plants, especially those propagated
vegetatively as bulbs or rhizomes may be virus
reservoirs (Flasinski et al., 1995) and often such
plants are kept in the same screenhouses as
seedlings of vegetable crops.
Like other preventive control measures, quar-
antine inspections may have the unwanted effect
of decreasing biodiversity. This was reported dur-
ing the stringent selection of virus-free accessions
of bean and pea (Alconero et al., 1985; Klein et
al., 1990). Moreover, the seed-borne virus itself
may decrease diversity by killing or greatly de-
creasing the growth of infected mother plants. In
considering threshold levels for seed-borne CMV,
loss of diversity could be an unavoidable cost to
be paid to ensure the introduction and subsequent
use of virus-free germplasm.
Sanitary certification is a natural complement
to quarantine measures for releasing virus-free
propagules to farmers. Voluntary certification of
tomato seedlings was launched in Apulia (south-
ern Italy) in agreement with Directive n. 33/92 of
the European Union, issued on 24 August 1992
and now enforced through national regulation.
Nurserymen entering the certification programme
must accept the technical requirements (green-
houses equipped with insect-proof nets and dou-
18 D. Gallitelli / Virus Research 71 (2000) 9–21
ble doors, weeds effectively controlled around the
glasshouses, insecticide sprays applied frequently
and periodic inspections by authorized personnel).
Phytosanitary certification requires the timely
and quick processing of many samples, as
achieved by a rapid and sensitive detection
method such as the multiple chemiluminescence
dot-blot procedure devised in the laboratory (Sal-
darelli et al., 1996).
7. Concluding remarks
This paper provides evidence of the ways in
which CMV outbreaks can impair the sustainabil-
ity of modern farming systems. The timely iden-
tification of new virus strains or reassortants
seems to be the most urgent need. However,
although reliable technologies have long been
available, this approach is still largely neglected
by extension services in many countries. In the
last decade, experience in countries of the Med-
iterranean basin is that initial infection of new
CMV strains in new host species may lead to
colonization of other susceptible species and un-
expected synergism with viruses already present.
The new strains may possess selective advantages
that may be further enhanced albeit inadvertently
by cropping practices introduced to improve pro-
ductivity. This quickly erodes economic benefits
and virus biodiversity and disrupts fragile natural
equilibria so that crop protection specialists have
had to face the challenge of devising new effective
and sustainable control measures.
Because it is now possible to genetically modify
plants (GMPs) to obtain CMV-resistant varieties,
the pathogen-derived resistance approach is gain-
ing momentum. However, do such GMPs affect
the sustainability of agriculture? The use of trans-
genic plants brings both benefits and risks, al-
though in current debates there is a preoccupation
with risks and little or no discussion of benefits. It
is believed that GMPs may provide very impor-
tant opportunities for improving the control of
viruses such as CMV that have a complex ecol-
ogy. However, there is no doubt that more effort
is needed, especially in the search for genes that, if
engineered into a crop species, may be less harm-
ful to the ecology of CMV than current practices.
Moreover, the large-scale use of GMPs must be
supplemented by other cropping practices and
periodic monitoring of CMV population for the
timely detection of adverse effects. These issues, if
properly addressed by Regional Extension Ser-
vices may benefit both agriculture and the socio-
economy by offering increased employment.
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