Celery and parsley plants that become infected in the early to middle part of their growing cycle are unmarketable while plants that become infected late may be marketable but with a reduced shelf life.
Celery mosaic virus (CeMV) causes a serious disease of celery (Apium graveolens
L.) It also infects other members of the plant family it belongs to (Apiaciae).
CeMV was first identified in South Australia in the 1980's but has now
spread throughout all worldwide (Alberts et al.,
1989). CeMV is transmitted by aphids in a non persistent manner (Severin
and Freitag, 1938) and Simons and Sylvester (1953).
It is a member of the genus Potyvirus and is restricted in host range to the
Apiaceae. CeMV is the most common viral disease of celery. CeMV infected plants
usually are stunted and have a flattened appearance. Leaflets often exhibit
a prominent mosaic, although sometimes only a faint mottle will appear. Leaflets
often are considerably more narrow than normal. Sometimes the leaflet margins
curl upward and raised areas occur on upper leaf surfaces. Foliage on some plants
is yellow or bronze. Petiole mottling frequently appears as light patches against
a darker background. CeMV affects only celery, carrot and closely related weeds.
CeMV is spread by aphids and by mechanical means (Latham
and Jones, 2003).
The virus is not reported to be seed borne; it is vectored by aphids. Celery
is the primary host of this disease. Celery mosaic virus (CeMV) is filamentous
with a length about 780 nm to 870 nm; width of 15 nm (Walkey
et al., 1970).
Hence, the aim of this study is to isolate and identify CeMV from celery and
parsley using host range and symtomatology, mode of transmission, enzyme-linked
immunosorbent assay examine and elucidate the effects of some plant species
for antiviral activity in form of oils of some medicinal plants on Chenopdium
MATERIALS AND METHODS
Virus source and symptoms: Samples of celery and parsley plants showing typical systemic mosaic, vein clearing, chloroatic spots and yellowing symptoms of CeMV were collected from different location in Mina governorate. The symptoms of CeMV in celery plants are severe vein clearing of leaves, leaf up-curling, leaf chlorosis and plant stunting.
Virus isolation and propagation: Diseased leaves of celery (Apium
graveolens L.) and parsley plants (Petroselinum crispum L.) showing
a mosaic symptoms suspected to be virus infections were collected from El-Mina
governorate. Detection of CeMV in the collected leaves samples was carried out
using polyclonal antibody as demonstrated by Clark and Adams
(1977) and Converse and Martin (1990). Antiserum
for CeMV was kindly provided by Diederichsen, Alex at Institute of Plant Genetics
and Crop Plant Research, Gatersleben/International Plant Research, Genetic resources
Institute, Rome, Italy.
The virus isolate was biologically purified through a single local lesion technique
repeated two times on Chenopodium amaranticolor CosteandReyn plants (Kahn
and Monroe, 1963; Kuhn, 1964). The virus was transmitted
mechanically to Nicotiana tabacum L. white burly for virus propagation
and used as a source of virus in the following experiments.
Mechanical transmission and host range: Different plant species belonging to 6 different families (Amaranthaceae, Apiaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae, Solanaceae) were mechanically inoculated with infectious sap in preliminary transmission tests; the transfer of CeMV from infected celery to herbaceous hosts was found to be satisfactory (1 g w/mL 0.01 M phosphate buffer pH 9.4, containing 0.001 M Na-DIECA).
Five seedlings of each host were inoculated and observed daily from symptoms development. A number of healthy seedlings of the same species and age were left without inoculation to serve as a control. Two weeks after inoculation, plants were visually examined for symptoms appearance and assays using serological techniques (DAS- ELISA test).
Insect transmission: Aphid transmissibility of the present viruses was
evaluated under controlled conditions on four weeks old (N. tabacum cv.
White Burly) seedlings. Colonies of aphid species used in tests were collected
in March and April, at the ARC (Horticulture Institute) Myzus persicae aphids
were collected from peach orchard from trees and associated herbaceous ground
cover in and around the orchard. Aphis gossypii were collected from a
bean field at the ARC. Also, in addition virus-free aphids were originally obtained
from the stock culture of Aphid Section, Plant Protection Research Institute,
Virus purification: The isolate virus was partially purified by a modification
of the method described by Shepard and Grogan (1967
and 1971) and Gamal El-Din et
al. (1997). Two hundred grams of frozen systemically infected tobacco
leaves were homogenized for 5 min in Braun blender with freshly 0.5 M potassium
phosphate buffer, pH 7.0 (1:2 W/V) containing 0.5% 2-mercaptoehanol, 0.5 M urea,
1% Na2So3 and 0.05 M sodium EDTA. The homogenate was squeezed
through two layers of cheesecloth and clarified with 7% n-butanol and 50% chloroform
clarification was undertaken by stirring the homogenate with chloroform, in
the blender for 5 min, then with n-butanol for 30 min at 4°C. The emulsion
was centrifuged at 8000 rpm for 15 min. The virus was precipitated from the
supernatant by adding 4% PEG and 3% NaCl slowly (w/v) with stirring for 30 min
at 4°C, then incubating the mixture overnight.
The virus was collected by centrifugation at 10000 rpm for 30 min and the pellets were resuspend in 0.1 M potassium phosphate buffer, pH 7.0, containing 1.0 M urea, 0.1% 2-mercaptoehanol and Triton X-100 (v/v) and with stirring overnight at 4°C. After a low speed centrifugation to remove insoluble material the virus was concentrated by one cycle of high speed 40,000 for 1.30 h, followed by 30% sucrose cushion 35,000 for 2 h.
Purified virus was layered onto 10-50% sucrose gradient columns, centrifuged
(50,000/1.30 h) using SW 60 rotor. Virus bands were collected from the sucrose
gradient using a syringe, diluted in 0.01 M potassium phosphate buffer, pH 7,
centrifuged (45,000 rpm/1.30 h/4 C), then resuspended in 0.01 M potassium phosphate
buffer. The virus was estimated spectrophotometrically using extinction coefficient
E0.1 1 cm 260 of 2.8 (Purcifull, 1990).
Electron microscopy: Virus preparation was examined with an electron microscope Model SEO TEM at the Electron Microscopy Unit, Military Veterinary Hospital, Cairo, Egypt after staining with 2% phosphotungstic acid (PTA).
Rabbit immunization and bleeding: A total 10 mg purified CeMV was used for subcutaneous injection. Purified virus was emulsified with an equal volume of Freunds incomplete adjuvant at weekly intervals. Rabbit was bleed 10, 15 days after the last injection.
Titer of antiserum: The titer of the induced antiserum was determined using indirect ELISA. Healthy and infected leaves of Nicotiana tabacum L. White Burly with CeMV were extracted (1:10 w/v) in coating buffer (0.05 M carbonate, 0.02% sodium azide, pH 9.6) at 1:10 (wt/vol) and clarified by centrifugation at 10,000 rpm for 1 min. The antigen was further diluted in coating buffer to 1:10 (vol/vol) for CeMV detection at 200 μL/well and incubated at 4°C overnight. After washing the plate, 200 μL I gG were added per well and plates were incubated at 4°C overnight. Plates were washed and alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma, A.8025) was added at a 1/7,000 dilution and incubated 2 h at 37°C. p-Nitrophenyl phosphate (200 μL/well) was added at 1 mg mL-1 and incubated at room temperature for 30 to 60 min for color development. Readings of reactions were carried out after 1 h at 405 nm in an ELISA Reader (ELx800 universal Microplate). Samples were considered positive when absorbance readings (A405) of sample wells were greater than twice times the mean value of three healthy controls.
Purification of immunoglobulin (IgG) from CeMV specific antiserum: Anti-CeMV
immunoglobulin was purified from the antiserum of CeMV according to the method
described by Brattey and Burns (1998). A mixture of 1
mL of crude antiserum and 9 mL of distilled water was added to an equal volume
of saturated ammonium sulfate, pH 7.0, while stirring. Contents were incubated
for 60 min at room temperature. The precipitate was collected by centrifugation
for 15 min at 9000 rpm and dissolved in 2 mL of half- strength PBS. The dissolved
precipitate was dialyzed 3 times against 500 mL phosphate buffer, pH 7.2 for
3 h/each to remove ammonium sulfate. The Optical Density (O.D) was measured
at a wavelength of 280 nm and the gamma globulin concentration was then adjusted
in half strength PBS to read 1.4 (1 mg mL-1). The resultant globulin
was stored at -20°C.
Incidence of celery mosaic virus in Apiaceae crops: Polyclonal antibodies specific to CeMV produced earlier was used in detected the virus in survey and evaluation the affective of mineral oils were used to inhibit the virus infection and also in Indirect ELISA assay (the secondary antibody, goat antirabbit conjugate with alkaline phosphatase sigma A4503) was used to detect the virus in the host plants. Leaf samples were collected from different farms in El-Minia and Kalubia Governorates.
Preparation of the inhibitor: The oils produced from of plant material
(Jasmine oil and Camphor oil) was mixing and shaking well in distilled water
(0.5:100 v/v) as antiviral inhibitor. These oils product were used as foliar
spraying 1, 2 and 24 h before virus inoculation to Ch. amaranticolar and
N. tabacum White Burly and were keeping under observation to record the
Serological test to detect inhibition: CeMV detected in apiaceae and
other different host range, samples were tested using conventional double antibody
sandwich enzyme-linked immunosorbent assay (DAS- ELISA) (Clark
and Adams, 1977), with coating IgG and IgG conjugates produced in this investigation.
Optical density (O.D.) was measured at = 405 nm in an ELISA micro well reader
(Dynatech Immunoassay MR 7000). Lyophilized samples obtained were used as positive
and negative controls in each ELISA plate. Positive threshold values were set
at twice average value for the negative controls (Clark and
Adams, 1977). Incidence levels were considered as all positive samples within
total number of samples for each province.
CeMV was detected in the inoculated and control plants by ELISA test after two weeks of virus inoculation.
Local lesion assay: The local lesion assay was performed on three plants
of Chenopodium amaranticolr for each treatment in greenhouse controlled
at 25±2°C. The third to sixth leaves from the top of the plant were
used. Carborandum of 600 meshes was sprinkled on the Chenopodium leaves
and CeMV inoculum was rubbed on the leaf surface with a small cotton ball. Local
lesions on the surface were counted after seven to ten days after inoculation
(numbers of local lesions per leaf then calculated the mean numbers of local
lesion) according to an inoculation scheme (Noordam, 1973).
The inhibition percentage of virus infectivity was calculated by the formula:
where, >C is average number of local lesion on control leaves Ch. amaranticolr.
T is average number of lesions leaves inoculated by virus inoculums with inhibitors
treated (Baranwal and Verma, 1992, 1997).
RESULTS AND DISCUSSION
Virus isolation and propagation: Virus isolate was obtained from naturally
infested celery and parsley plants showing mosaic and malformed leaves (Fig.
1 a, b and d) collected
from three commercial farms in Minia Governorate. After successive single lesion
transferred in C .quinoa, the resulting virus isolate was propagated
in N. tabacum var. White Burley. The symptoms were very similar to those
illustrated by Hollings (1964) and Alberts
et al. (1989). Subsequent work clearly proved that the virus under
study is Celery mosaic virus. These results were based mainly on symptomatology,
host range, modes of transmission and serology.
||Symptoms of CeMV on naturally and artificially infected host
plants; (a) Mosaic, chlorotic spots and deformation appeared on naturally
infected celery, (b) CeMV symptoms (mosaic and browning) appeared on artificially
infected celery leaves, (c) Symptoms on dill plants artificially infected
showing yellow leaflets, (d) Mosaic and chlorotic on artificially infected
parsley plants, (e) Mosaic and yellow symptoms on coriander artificially
infected leaves, (f) Mosaic and deformation in carrot leaves infected with
CeMV, (g) Chlorotic spots in C. quinoa CeMV infected leaves, (h)
Chlorotic local lesions appeared on Ch. amaranticolor, (i) Symptoms
on infected N. tabacum var. White Burley showing sever mosaic and
deformation, (j) N. rustica reacted the severe mosaic after infected
with CeMV, (k) pepper artificially infected showing mosaic and epinasty
and (l) Electron micrographs of purified CeMV showing filamentous; flexuous
shaped virus particles stained with 2% uranyle acetate (40,000)
|| Symptomatological responses of herbaceous host ranges to
CeMV isolated from Celery
|B = Blisters, CS = Chlorotic Spots, Cu = Curling, Co = Chlorotic,
VB = Vein Banding, VC = Vein Clearing, LD = Leaf Deformation, M = Mosaic,
MN = Marginal leaflet Necrosis, St = Stunting, D = Death of plant, LL =
Local Lesion, MM = Mild Mosaic, Y = Yellow, W = Wilting, (+) Positive reaction,
(-) Negative reaction
Mechanical transmission and host range: Celery is most common host of this virus. As the name implies this virus causes a mosaic or mottling in the leaves of celery. There can also be malformation of leaflets. In order leaves chlorotic or necrotic spots may occur and the plants can be stunted. In addition to celery, this virus has been identified in other important crops in Apiaceae including carrots (Daucus carota), coriander (Coriandrum sativus), parsely (Petroselinm crisum), dill (Anethum graveolens) and Anise (Pimpinella anisum).
Results of host range trials showed that CeMV had a wide host range (Table
1, Fig. 1). Seventeen plants of twenty four plant species
and cultivars belonging to six families were susceptible with CeMV and many
of them were infected systemically showing different symptoms. No symptomless
infection was detected by DAS-ELISA in leaves (Table 1). CeMV
locally infected C. quinoa and C. amaranticolor (Fig.
1g, h) In C. quinoa irregular chlorotic spots on
the inoculated leaves were observed 7 days to 10 days after the inoculation
These results showed that distinct virus is associated with mosaic, chlorotic rings and curling in different hosts. The virus was identified as Celery mosaic virus (CeMV).
Insect transmission: Experiments demonstrated that Aphis gossypii
and Muzus persicae were able to transmit CeMV in non persistent manner
from infected celery plants to the same healthy celery or parsley, carrot, coriander
and N. tabacum, M. persicae was more effective than A. gossypii
in transmitting CeMV. Data in (Table 3) revealed that
Aphis gossypii and Muzus persicae were able to transmit CeMV from
infected celery plants to healthy celery, parsley, carrot, coriander and N.
tabacum. These results were agree with Purcifull and
Shepard (1967), Zltter (1970) and Latham
and Jones (2003).
Virus purification: Using the purification method one band was observed,
1.5 cm below the meniscus in the sucrose density gradient columns. The ultraviolet
absorption spectrum of CeMV was characteristic of a nucleoprotein, had a maximum
absorbance at 260 nm, a minimum absorbance at 243 nm and A260/280 ratio of 1.12
and Amax/min ratio was 0.96. These results are similar to that estimated by
Brunt et al. (1996). These results suggested that
the purification method was quite successful in purifying the virus under study.
Virus yield was 6.7 mg/100 g of infected tissue. Matthews
(1991) reported yields for Potyvirus from different laboratories
may vary quite widely due to such factors as host species, growing conditions
and isolation procedure.
Electron microscope: Electron microscopic micrograph of the purified
preparation revealed the presence of filamentous particles with average length
of 700-780 nm and (Fig. 1l). These results are similar with
Walkey et al. (1970) and Brnut
et al. (1996).
Titer of CeMV antiserum: Titers of the induced antiserum measured with
indirect ELISA were 17500 from bleeding obtained after the first and 1/7000
in the second one which is an indication of the high immunogenecity of this
antiserum (Table 2). Antiserum obtained after the first bleeding
(1:7500) was used in the subsequent experiments.
|| Determination of CeMV antiserum titer in relation to time
of blood collection
||Results of transmission test CMV and PPV by aphids from apricot
to apricot, peach
|+: Virus was transmitted, -: Virus was not transmitted
|| Effect of foliar spraying with Jasmine oil (0.5 %)
and Camphor oil (0.5 %) of certain medicinal oils on celery mosaic
virus infected N. tabacum cv. White Burly (as a systemic host) and
Ch. amanticolor (as Local lesion host)
Matthews (1991) obtained that the antiserum to be used
for virus estimation are best produced in rabbits, from which a large volume
of serum. It is worth to try to produce an antiserum of high titer. This allows
high dilution of the serum, thus avoiding nonspecific inhibitory effects of
serum at low dilution and allowing more tests to be made from a given volume
Purification and conjugation of IgG with alkaline phosphatase: Purification
of IgG was done according to the procedure described by Brattey
and Burns (1998). The concentration of IgG was adjusted to 1 mg mL-1
Incidence of celery mosaic virus in Apiaceae crops: From 2008
to 2010, surveys were carried out to determined the incidence of CeMV in carrot,
celery, coriander and parsley crops in El-Minia and Kalubia Governorats. CeMV
was found on 75% in celery, 72% in carrot, 40% in parsley and 35% in coriander
samples. In this investigation Latham and Jones (2003)
determined the incidence of CeMV in celery crops in South-west Australia, CeMV
was found in all farms samled in this region. Incidences of infection in individual
crops were 43-96%.
Virus inhibition: CeMV is easily transmitted by mechanically inoculation.
Also, in nature, it is transmitted by aphids in a non-persistent manner. Once
the virus is detected in the field, the major control method is to immediately
eradicate plant materials which developed symptoms (Zltter,
When study the effect of some oils medicinal plant species (Jasmine and Camphor), for antiviral activity in the form of foliar spraying. Data in Table 4 revealed that the O.D. values determined by ELISA reader (indicate CeMV concentration in N. tabacum White Burly) was 0.421 with jasmine oil treated, 0.668 with camphor oil treated and 1.114 for untreated-virus infection leaf samples CeMV infectivity was determined by counting the average number of local lesions (6 leaves were used in each trail) subsequently formed after 6 and 7 days of back inoculation on Chenopodium amaranticolr and then switched to percentage the inhabitation and reduction virus infection were: Jasmine oil 80% and 76.5% ; camphor oil 74% and 78.2%, 55, respectively.
These results agree with studies of Milbrath and Ryan (1938).
Peters and Lebbink (1973). Sugimura
et al. (1995), DAntonio et al. (2001)
and Shoresh et al. (2005). Also, they motin that
yet, there were no efficient chemical treatments that protect plants from virus