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Everglades Station Mimeo Report EES 70-7 May, 1970
Behavior of Viruses in Celery
T.A. Zitter 1/
The two virus diseases affecting celery, cucumber mosaic virus (CMV)
and western celery mosaic virus (WCMV), were studied in detail during the
past year. Since both are stylet-borne,aphid-transmitted viruses, knowledge
of their behavior in susceptible plants will help to explain spread under
field conditions. Attention was given to the ease of infection, symptom
development and recovery of the viruses from infected plants. Virus
multiplication in single and mixed infection was studied to determine how
virus concentrations correlate with aphid acquisition and transmission.
Investigations were conducted to determine if mechanical injury resulting
from topping plays a role in virus spread. The role of celery as the
source of inoculum for field spread of western celery mosaic virus is
MATERIALS AND METHODS
All experimental work was done in the lab and greenhouse unless
mentioned otherwise. The cucumber mosaic virus isolate used in this
study was originally isolated from Commelina sp. and was maintained in
either tobacco or celery plants. Field isolates of western celery mosaic
virus obtained in 1969 were continually maintained in celery for further
investigation. Young leaves from plants infected for 2 to 3 weeks were
used as sources of inoculum.
Mechanical inoculations.- Celery plants used in most of these experiments
were mechanically inoculated by the following procedure. Crude sap was
prepared by macerating CMV or WCMV infected leaf tissue in a mortar with
0.01M PO4 buffer, pH 7.0 (1:10 W/V) with the addition of charcoal during
grinding. The latter step was essential for good transmission of CMV. When
celery plants were mix-infected with both viruses simultaneously, equal
amounts of infected tissue were macerated together. The inoculum was
passed through four thicknesses of cheesecloth and collected in a Petri
dish. Celery plants in the two leaf-stalk stage and with the heart
leaves just beginning to unfold, were lightly dusted with 600 mesh
carborundum. Several leaflets on two tagged leaf-stalks were then
rubbed with a sterile glass spatula dipped in the infectious sap. Inoculated
plants along with healthy controls were placed in the greenhouse for
observation. Plants were checked at weekly intervals for symptom
1/ Assistant Professor (Assistant Plant Pathologist), Everglades Experiment
Station, Belle Glade, Florida.
appearance, and subsequently formed leaf-stalks were tagged. Pooled
virus samples from infected plants were collected, weighed, and then
frozen for future assay. CMV titers were determined by local lesion
assay on half leaves of Chenopodium amaranticolor Coste and Reyn
(lambsquarters). Each treatment was randomized on 12 leaves, giving
24 half leaves for comparison. Lesions appeared 2 days after inoculation
and were counted 2-3 days later. Western celery mosaic infections were
followed by symptom appearance and were confirmed by microscopic
observation of cytoplasmic inclusion bodies. Thin epidermal strips of
infected celery leaflets were stained and mounted on glass slides for
Mechanical topping.- In order to test the possibility of virus spread by
clippings ofcelery seedlings, the following experiment was performed.
Celery plants of either Florida 683 or FFPE 2-14 were grown in bagasse
boards and flats, respectively. The dense population of plants grown
under these conditions offered a situation similar to that found in
celery seedbeds. Western celery mosaic was selected for this test
because of its characteristic symptoms and its proven ease of transmission
by mechanical rubbing. Preparation of virus inoculum was essentially the
same as described above. Scissors contaminated by being dipped directly
into infectious sap were used to top celery to a height of 4-5 in. In
an additional treatment, a row of celery plants (5-6 plants) was mechanically
inoculated by rubbing with a glass spatula. After symptoms appeared,
each row of plants was topped with a scissors, starting with the infected
plants and working backward. Results were recorded after a 3-4 week
Aphid transmissibility.- Only limited work was done using this approach.
The aphids used were Myzus persicae :Sulzer and Aphiscraccivora
Koch, which were reared under controlled conditions on virus.'free plants
of pepper (Capsicum annuum L. cv. Early Calwonder) and cowpea (Vigna
sinensis L. cv. Black Local), respectively. Nonviruliferous aphids
were starved for 1-6 hours and were transferred to leaves of virus
source plants. Aphids were viewed with a binocular microscope and
probes were not allowed to exceed 1 minute, After probing, the aphids
were transferred to healthy test seedlings (celery in the case of WCMV and
pepper as the test host for CMV), and were killed 24 hours later with
either nicotine sulfate or malathion.
Mechanical inoculations.- Factors affecting mechanical transmission of
CMV and WCMV to celery include age of test plants, virus concentration
in source tissue, and proper preparation of inoculum. This is especially
true for CMV where erratic and variable results can occur. Consistent
results were obtained in the case of WCM with 100% infection in all cases.
All commercially grown celery varieties are susceptible to both viruses.
However, differences in susceptibility have been noted. In addition, two
foreign celery lines selected for bacterial blight resistance were moderately
susceptible to both viruses. In one test conducted under controlled
conditions in growth chambers, the average incubation period in days for
CMV in selected celery varieties was as follows: FFPE 2-14, 10; Florida 683,
12; June-Belle, 13; and Earlibelle, 15. The greater susceptibility of
2-14, allowing for more rapid systemic movement and virus multiplication,
has direct implications on the amount of spread in the field. The
percent infection of 2-14 celery resulting from aphid transmissions has
been found to be 3-6 times greater than that of 683 celery.
Symptomatic expression of CMV in celery is essentially the same for
all varieties, but can be best illustrated in the case of 2-14 celery.
When leaf-stalks 1 and 2 are mechanically inoculated with CMV, only limited
multiplication occurs and little virus is recovered from these leaflets.
Systemic movement of the virus proceeds quickly from these leaf-stalks to
the young developing heart leaves where the first virus symptoms appear as
vein clearing and puckering. This usually occurs 8-10 days after
inoculation. If the 3rd leaf-stalk was already developed at the time of
inocllation, the systemic flow of virus proceeds directly to the youngest
tissue (4th leaf-stalk). The inoculated leaf-stalks develop symptoms
3-4 weeks after inoculation, consisting of yellowing, necrotic specks,
and sunken lesions on the petioles. Depending on the developmental stage
of leaf-stalk 3 at the time of inoculation, it may show necrotic specks
or remain symptomless. Initial symptoms are rapidly replaced by
mottling and later by necrotic and chlorotic specks along the veins and leaf
blades and this is repeated as each successive leaf-stalk develops.
Necrotic breakdown of the veins appears as oak-leaf patterns 5 to 6 weeks
after inoculation. Virus recovery is possible from all above ground parts
including the sunken spots on celery petiole. However, age, size of
plants, and time of infection will determine how much virus is recovered.
Although the most active virus multiplication occurs in young tissue showing
faint symptoms, quantitatively more virus is recovered from older
systemically invaded tissue 1-2 weeks later, when good chlorotic and
necrotic symptoms are present.
Multiplication of CMV in single and mixed infections with WCMV.- FFPE 2-14
celery was selected as the test host for CMV multiplication studies
because of its proven susceptibility. The multiplication of CMV in
single and double infections was followed by local lesion assay on
lambsquarters as described. This host proved to be more reliable than
cowpea used in earlier studies. Celery plants were also singly infected
with WCMV for comparison.
Very limited multiplication of CMV in singly infected plants occurred
in the two inoculated leaf-stalks one week after inoculation. A
pronounced peak of virus activity occurred in the third leaf-stalk 2
weeks after inoculation. Virus titer dropped sharply between the third
and fifth week when leaf-stalks 4 through 6 were sampled, and little if any
virus was recovered. This was followed by a gradual rise in virus titer
between the sixth and eighth week after inoculation, approaching the level
of activity at the 2-week assay. In plants infected with both viruses,
CMV multiplication followed essentially the same pattern with one exception.
The peak of virus activity was delayed one week, and occurred in the fourth
rather than the third leaf-stalk. Plants doubly infected were severely
stunted and heavily mottled compared with 2-14 plants singly infected with
either CMV or WCMV. Surprisingly though, the rate of development
(number of tagged leaf-stalks) was essentially the same in all three groups.
Plants infected with CMV showed very mild symptoms on the young leaflets,
and could be mistaken as healthy were it not for the presence of necrotic
specks and patterns. On the other hand, WCMV infected plants showed
good mosaic symptoms throughout the experiment.
Mechanical topping.- The results for attempted infection and spread of
tW(~6y-mechanical topping are summarized in Table 1. Only when special
steps were taken in the preparation of virus inoculum (treatment 1) was
it possible to effect successful transmission. Inoculum was prepared
from celery plants infected for 3-4 weeks, thus insuring a very high
virus content. Abundant virus was therefore available for uptake when
the celery petioles were injured with the scissors. These factors do not
exist in commercial celery seedbeds and thus discredit the possible
spread by topping. Treatment 3, which closely approaches the conditions
which could exist in seedbeds, gave negative results. Low virus titers
and possibly the instability of the virus could account for this response.
Since the greater susceptibility of 2-14 celery was again shown with this
method of inoculation, further testing using this variety should be made.
Table 1.- Attempted infection and spread of WCMV by mechanical
Experiment 1 2
1. Scissors contaminated
by dipping into infec-
2. Same as treatment 1
3. One row of seedlings
inoculated and then
4. Scissors contaminated
as in treatment 1,
surface sterilized with
5. Control-sterile scissors
used for topping
Same as for 1
buffer and charcoal
infection in 12
Same as treatment 1
Experiment 2 3
Same as for 1
1/ Reduction in number due to removal of infected
plants or normal plant
2/ Florida 683 celery grown in bagasse boards. Each treatment replicated
3/ FFPE 2-14 celery grown in wooden flats. Two replicates only.
Aphid transmissibility.- An aphid's ability to acquire and transmit virus
is sti-ongly dependent on the titer of virus in source tissue. The titer
of CMV in celery has been shown by local lesion assay to fluctuate
depending on the time after inoculation. Preliminary experiments using
aphids to assess CMV titers have shown similar results. Greatest aphid
transmission corresponds to the peak of virus activity and decreases
as the virus titers decline. Selecting CMV infected tissue solely on the
basis of good symptom appearance has usually resulted in poor aphid
transmission. Generally speaking, aphid transmission of WCMV has been
much higher than with CMV. Selecting older WCMV infected tissue as the
virus source and using 5 aphids per test plant has given as high as 50%
transmission, whereas comparable CMV infected tissue gave negative results.
Similarly, in celery naturally infected with both viruses, only WCM was
acquired in one trial made. The greater ease of transmission of WCM
by aphids will be studied further, and may explain the faster and more
extensive spread of this virus in the field.
Field spread of Western Celery Mosaic Virus.- Essentially no WCMV occurred
in commercial fields this spring; however, this virus did appear in plots
established at the Everglades Experiment Station and thus provided an
opportunity to study field spread of this disease.
Cucumber mosaic virus appeared early in celery breeding plots and
resulted in substantial plant loss due to extensive spread of this
virus. WCMV appeared much later and was first confirmed in these plots on
December 22, 1969. Both viruses were more or less restricted to the
west end of these plots, with only scattered CMV infected plants found
elsewhere. Only occasional WCM infected plants were found and then
usually as a mixture with CMV. It is worthwhile to note that although
aphid flights were heavy at this time, the spread of WCM was very low
compared to its behavior during spring months. Old celery plants saved from
the previous season and planted in a row bordering the northwest side of
the plots were strongly infected and undoubtedly were the source of inoculum
for both viruses. Scattered patches of dollarspot (Hydrocotyle) were
found along the entire length of a bordering ditchbank. However, no
infected plants were found and because the restricted appearance of
WCM correlated closely with the old celery plants, it is doubtful that
weeds served as an inoculum source. The infected celery plants in these
plots were disked under in late January.
On December 19, additional celery plots were established by Entomology,
southeast of the original plots and at a distance of approximately 1500
ft. Celery plants for this experiment were grown in a greenhouse and
transferred to a shade house prior to field setting. This treatment
helps to insure their freedom from virus. In addition, plants handled in
the same way and obtained at this time for greenhouse studies failed to
develop any symptoms during several months of observation.
No insecticidal sprays were applied to these plots before April 14,
in order to allow time for buildup of insect populations and subsequent
evaluation of insecticides. Infected plants showing symptoms typical for
western celery mosaic were first found in March. By the middle of April
tlis virus had spread throughout the entire plot area. Very few
cucumber mosaic infected plants were found. This was the direct reverse
of the situation that existed in the fall planting.
The preceding discussion offers a vivid account of virus spread. It
emphasizes the importance of celery as the primary and secondary source
of inoculum. Overlapping of the two plantings offers a sound explanation
for the source of WCM that appeared in the later planting. Source of
inoculum, timing, distance and presence of vectors combined to insure
subsequent virus spread. The predominance of CMV and limited spread of
WCM in the fall coupled with extensive and rapid spread of WCM compared
with CMV in the spring, indicates that host-vector-virus relationships
uist play an important part in the spread of both viruses. Further
investigation are needed to explain this behavior.
The multiplication of CMV in celery fits the pattern of this virus
in other crops such as beans and peppers. Maximum virus concentrations
are reached soon after inoculation and then decrease rapidly. If virus
source tissue is selected after peak activity occurs, very little virus is
available for subsequent transmission. This serves to explain why
erratic results have occurred when celery plants were mechanically
inoculated. It also suggests why CMV spread in the field is more localized.
WCMV titers remain more constant in celery. This enables aphid vectors
to acquire virus over a longer period of time and can account for more
extensive spread in the field. The fact that WCMV is usually recovered
from mix infected plants can also be explained on this basis.