Chrysanthemum is a genus of about 30 species of perennial flowering
plants in the family Asteraceae. Chrysanthemum is one of the three most
important cut flowers in the world. Chrysanthemum has an economical importance
as ornamental, culinary, environmental and insecticidal uses (Marongiu
et al., 2009). Chrysanthemum cinerariifolium is economically
important as a natural source of insecticide with a huge international market
(Liu and Gao, 2007) Chrysanthemum plants are
infected with viruses as Tomato Aspermy Virus (TAV) (Verma
et al., 2006), Tobacco mosaic virus (TMV) (Zaitlin
and Israel, 1975) and Cucumber Mosaic Virus (CMV), Chrysanthemum
B Virus (CVB) (Ram et al., 2009) and viroids
as Chrysanthemum Stunt Viroid (CSVd) and Chrysanthemum chlorotic
mottle viroid (CChMVd) (Hosokawa et al., 2005).
The current study aims at identifying and characterizing an Egyptian TMV isolate from naturally infected Chrysanthemum cinerariifolium based on its biological, serological, and molecular properties and the production of virus free plant materials.
MATERIALS AND METHODS
Source of the virus isolate: About 35 Chrysanthemum plants were collected from garden and houses plants of Horticulture Dept. Fac. of Agriculture, Ain Shams University. Egypt. Some of these samples showed virus like symptoms.
Biological properties of isolated: Infected Chrysanthemum plants with foliar symptoms were used in mechanical inoculation of indicator N. glutinosa plants. Infected Chrysanthemum tissues were triturated (1:1, v/v) with 0.05 M phosphate buffer pH 7.2, containing 0.2% mercaptoethanol. Homologous single local lesion produced on N. glutinosa were cut and macerated on a glass slide and mechanically inoculated on seedlings of N. tabacum cv. Samsun as a propagative host.
Infected sap from Samsun tobacco was used in host range testing using (20 plant species belonging to 7 families). Infectivity of inoculated plants was tested with ELISA (Table 1).
Physical properties characters as Thermal Inactivation Point (TIP), Dilution-End
Point (DEP) and Longevity In Vitro (LIV) of the TMV isolate was determined according
to Noordam (1973), using Ch. Aum production amaranticolor
as a local lesion assay host.
The virus was purified from inoculated Chrysanthemum plants according
to El-Ahdal et al. (1984).
Antiserum production: An adult New Zealand rabbit was injected with
TMV purified preparations seven times at one week intervals according to the
method of Makkouk and Gumpf (1976). The titer of the
antiserum was determined by indirect ELISA technique (Dijkstra
and DeJager, 1998).
Molecular characterization of the TMV isolate: Total RNA was extracted from healthy and infected Chrysanthemum plants using High Pure RNA Tissue Kit (Roche Molecular Biochemicals, Cat. No.2033674).
RT-PCR protocol was as described by (Chung et al.,
2007) using two primers (TMV1- ATGTCTTACAGTATCACTACTCC/ and TMV2-TCAAGTTGCAGGACCAGAGG
for the coat protein gene. Complementary DNA (cDNA) synthesis was accomplished
as described by Chung et al. (2005). Amplification
of the cDNA included the following cycles: Denaturation at 94°C for 30 sec,
primer annealing at 50°C for 1 min and extension at 72°C for 1 min for
40 cycles with a final extension at 72°C for 7 min. The PCR products were
analyzed by electrophoresis onto 1.0% agarose gel and the size of the DNA fragment
was determined in accordance with the molecular weight markers.
||Occurrence of TMV in infected Chrysanthemum samples
using produced antiserum and common antiserum
|Negative control for Chrysanthemum = 0.106; Positive
control for Chrysanthemum = 0.212
The PCR products were visualized on a UV transilluminator (λ = 254) and
photographed with an UVB laboratory products, Epichemi II Dark room, 3UV transilluminator
DNA sequencing for the partially amplified coat protein gene (~600 nt) of TMV
isolate was performed using ABI prism 3100, Genetic Analyzer by using dye- primer
and dye terminator method at Gene Link D) NA Sequencing service, New York, USA.
The resulting sequence for CP gene was then compared to the published sequences
of TMV using the Clustal W method (Thompson et al.,
Production of Chrysanthemum
TMV-free plantlets: Media used in this investigation was salt mix of
Murashige and Skoog (1962). The infected explants were
aseptically cut into single nodal pieces. The base of each cutting was cultured
in jars contained 25 mL of MS supplemented with 0.25 mg L-1 BA and
at 25±1°C under florescent light. The heat treatment was applied
to the in vitro micropagated plant material Shoot proliferation were
pruned and placed in growth chamber at 37±1°C for 14 and 28 days
at 16 photoperiod .Three different concentration of 10, 20 and 30 mg L-1
of filter sterilized virazole were tested. Combination between thermotherapy
and chemotherapy were applied in vitro by subjecting the micropropagated
shoots at 37±1°C for 21 days and then transferred to media containing
10, 20 and 30 mg L-1 virazole. After 2 weeks, shoots were placed
in antiviral free media. A period of about 35 days was necessary to have all
the cultures well- established and the shoots proliferated: then a few leaves
could be sampled for virus detection using produced antiserum by indirect ELISA
Symptomatology and hostrange studies: Naturally infected Chrysanthemum
plants exhibiting symptoms of conspicuous mottling, mosaic and vein banding
of leaves are shown in Fig. 1.
Host range studies showed the formation of systemic mottling, mosaic and blisters upon virus inoculation on N. tabacum cv. Samsun. Whereas chlorotic local lesions surrounded by necrotic tissues were developed on inoculated N. glutinosa (Fig. 2).
According to the response of the tested hostrange plants (Table 1), host reactions was classified into four categories as follows: 1) Plants showed local infection including Cenopodium quinoa, C. murale, Vicia faba cv. Giza 402 and C. amaranticolor, Gomphrena globosa, N. glutinosa and N. tabacum cv. White Burley, Datora metel and D. stramonium, 2) Plants showed different systemic symptoms including, N. tabacum cv. Samson, L. esculentum cv. Castle rock, Capsicum annuum, Jasminum mesnyi, Jasmonium multiflorum, J. mesnyi, J. grandiflora and Chrysanthemum spp. 3) Plants showed local lesion followed by systemic symptoms including, N. gluca and N. rustica and 4) Plants showed no visible reaction with TMV isolate included, Cucarbita maxima, Cucarbita sativus and Plumeria sp. These results were confirmed by indirect ELISA.
Inclusion bodies: Light microscopy of the epidermal strips from infected
Chrysanthemum morifolium leaves, 21 days post TMV inoculation showed
cytoplasmic inclusion bodies. The crystalline inclusions induced by TMV were
observed in epidermal and hair cell as well as amorphous inclusions stained
with bromophenol blue and mercuric chloride. In addition, the virus infection
caused decrease in number of stomata and lignifications of stomata gap as well
as irregular of epidermal cells compared with healthy (Fig. 3).
||Different types of symptoms on naturally infected Chrysanthemum
morifolium. (A), Healthy Chrysanthemum, (b) Leaves showing vein
banding, (c) Flower showing color breaking and (d) Leaf curling with vein
||Differential hosts mechanically inoculated with TMV isolate
showing: (a) Necrotic local lesions with chlorotic spots on N. glutinosa
and (b) Mosaic and mottling on N. tabacum cv. Samasun
Production of antiserum: The titer of induced antiserum was determined
by indirect ELISA was 1/1024. The induced antiserum for the TMV-Ch-EG isolate
(AS1) was more specific than commercial authentication serum (AS2) in detection
of TMV in Chrysanthemum samples (Table 1) and other
hosts (Table 2). Molecular characters.
||Epidermal cells of Chrysanthemum leaves inoculated
with TMV-Ch-EG isolate showing. (a) Crystalline (CI) and (b) amorphous (AI)
||1.0% Agarose gel electrophoresis showing partially amplified
PCR products for the TMV/CP gene. lane at the left represents 1 kb DNA markers;
lane 1, inoculated Chrysanthemum morifolium, lane 2 and 3: naturally
infected Chrysanthemum morifolium
||Occurrence of TMV in infected Chrysanthemum samples
using produced antiserum and common antiserum
|Negative control = 0.105 nm
RT-PCR: RT-PCR successfully detected TMV viral RNA from Chrysanthemum
tissues. Partial amplification of the CP gene about 600 bp in size was detected
from TMV infected tissues by using TMV1 and TMV2 specific primer for the coat
protein gene (Fig. 4).
||The partial nucleotide sequence (598 nt) analysis of the TMV/CP-gene
for the TMV-Ch-EG isolate
||Phylogenetic tree constructed from the multiple alignments
of the CP/TMV-Ch-EG (GeneBank accession GU982315) and six TMV strains
(AY686725_Egypt, AB369276_ Korea, AJ239099_China, AF273221_USA, AJ429078_
Germany and AY633749_ Thailand)
Results for the partially amplified DNA sequence of the CP gene is presented
in Fig. 5.
Phylogenetic analysis: The partial nucleotide sequence (598 nt) of the TMV/CP-EG was aligned with six coat protein sequences of TMV (Fig. 6) by using DNAMAN programme (DNAMAN V 5.2.9 package, Madison, Wisconsin, USA). A phylogenetic tree of TMV/CP-EG showed 89% sequence similarity with the other compared accessions.
Deduced amino acids sequence of TMV/CP gene: Deduced amino acids were determined using computer software. The predicted number of amino acids produced from translation of partial CP gene nucleotide sequence of TMV-Ch-EG (GeneBank accession ADF47092.1) isolate was 159 amino acids starting with Methionine (M) and ended with Serine (Fig. 7).
The predicted amino acids sequence (159 amino acids) of the TMV-Ch-EG CP-gene
was published in GeneBank accsseion ADF47092.1 and compared with other nine
amino acid sequences of TMV published in GeneBank.
||Predicted amino acids of partial CP gene of sequence of TMV-Ch-EG
isolate (GeneBank accsseion ADF47092.1)
||A multiple alignment of predicted amino acid sequences encoding
the Coat Protein (CP) gene of TMV-Ch-EG isolate (GeneBank accession ADF47092.1)
A phylogenetic tree of TMV-EG presented in (Fig. 8) revealed
that the deduced amino acid sequence of TMV under study has 82% resemblance
accessions (AAK33120_Brazil, AAT45114_Thailand, P69507_Japan, AAK06750_Korea,
AM64218_China, AAQ12020_Finland, CAL69972_Vietnam, ACY412-14_USA and CAD22079
Comparisons between the predicted amino acids of partial CP-gene sequence of TMV-Ch-EG isolate were showed that, the type of amino acids consists of 18 amino acids. Threonine and (Serine and Leucine) have a high frequency and percentage 17 and 10.69%, respectively, when Histidine and Methionine have low frequency and percentage 1 and 0.62%, respectively (Table 3).
Production of virus free plantlets using tissue culture techniques: Combining chemotherapy and thermotherapy by subjecting the infected shoots to 3^°C for 2 weeks in addition to 10, 20 and 30 mg L-1 virazole produced ratios of 95, 90 and 85% Chrysanthemum survival, respectively, while the percentages of virus free were 38, 61.5 and 76.8%, respectively (Table 4).
||Composition of predicted amino acids of partial cp gene sequence
for TMV- Ch-EG isolate
|A.A.: Amino acids
||The effects of thermotherapy and chemotherapy (virazole) on
TMV elimination from infected C. morifolium plants
||Micropropagation stage of Chrysanthemum plants in
vitro (healthy and infected). (1) 1-Starting stage; (a) Healthy and
(b) Infected. (2) Multiplication stage (a) Healthy, (b) Infected and (3)
Rooting stage (a) Healthy (b) Infected
||Effect of temperature (thermotherapy) on TMV infected C.
morifolium plants (a) 2 weeks and (b) 3 weeks
||Effect of Virazol on TMV infected C. morifolium plants.
(a) mg L-1, (b) mg L-1 and (c) mg L-1
Micro propagation stage of in vitro Chrysanthemum plants healthy and
infected are shown in Fig. 9. while the effect of thermotherapy
and chemotherapy are shown in Fig. 10 and 11,
According to biological and molecular characters, TMV isolated from Chrysanthemum (TMV-Ch-EG) is considered as a strain of TMV common (isolated from tobacco plants).
TMV isolate used in this study was found to give necrotic local lesions on
N. tabacum cv. White Burley plants and chlorotic local lesions surrounded
with necrotic tissue on N. glutinosa. These findings differed from that
obtained by (El-Ahdal et al., 1984; El-Afifi
et al., 2003) who described severe mosaic and malformation on N.
tabacum cv. White Burley and necrotic local lesions on N. glutinosa.
The partial nucleotide sequence of the RT-PCR amplified fragment with TMV1 and TMV2 primers for the coat protein cp-gene of TMV isolate was done to determine the relationship with other recommended TMV strains registered in Gene Bank.
Threonine and (Serine and Leucine) have a high frequency and percentage 17
and 10.69% respectively, when Histidine and Methionine have low frequency and
percentage 1 and 0.62%, respectively (Chung et al.,
A phylogenetic tree of TMV isolate revealed 89% a moderate degree of similarity
to the other isolate sequences of TMV strains while that obtained by Cherian
et al. (1999), TMV (Tom-K) nucleotide sequence of coat protein cp-gene
showed 93.1% identity with other TMV strains and by Chung
et al. (2007) nucleotide sequence of coat protein cp-gene of TMV-pet
showed 99.0% identity with TMV-potato 3-2 (GeneBank accession no. AF318215)
isolated from potato showing yellow mosaic and stunt symptom and with a TMV
Korean strain (GeneBank accession no. X68110).
The predicted number of amino acids was produced from translation of partial cp-gene nucleotide sequence were 159 amino acids starting with Methionine (M) and ended with Serine (S). Comparison between amino acids composition of partial cp-gene sequence for TMV isolate and nine TMV strains published in GeneBank (accessions (AAK33120_Brazil, AAT45114_Thail-and P69507_Japan, AAK06750_Korea, AAM64218_China, AAQ12020_Finl and, CAL69972_Vietnam, AC- Y41214_USA and CAD22079_Germany) was done to determine amino acid composition and revealed 82% aminoacids similarity with these TMV strains.
The biological, serological and molecular characters reported in this study confirmed identification of an isolate TMV-Ch-EG strain of TMV Tobamovirus isolated from C. morifolium in Egypt.
In our results, shoot tips (explants) were cultivated after sterilization process
in media containing 0.25 mg L-1 BAP that mainly resulted in the growth
of stem, leaves and occurrence of auxiliary shoots. While the micropropagated
stage was done by sub cutting of the stem cuttings up to two times every 21
days. These results were in agreement with Liu and Gao (2007)
who mentioned that rapid propagation technology was established and optimized
in vitro on MS medium supplemented with 0.3-0.5 mg L-1 benzyl adenine
(BA) and 0.3 mg L-1 Naphthalene Acetic Acid (NAA). Root induction
and development could be observed within 15 days after inoculation on 1/2 MS
medium supplemented with 0.2 mg L-1 Indole-3-Acetic Acid (IAA). Subculture
of these shoots on the same medium resulted in similar responses in multiple
shoot formation within 4 weeks. Also, the results of Patil
et al. (2005) revealed that the shoot tip explants of Chrysanthemum
inoculated in MS basal media containing 2 mg BAP and 0.5 mg NAA/L gave better
performance in terms of response to shoot initiation, the days to shoot initiation
and number of shoots per culture. The best rooting was obtained in MS basal
1.0 mg NAA/L.
Therapeutic treatments were tested since the 1920s to cure valuable cultivars from virus-like diseases.
The obtained results indicated that during in vitro heat therapy at
37°C±1 for 14 and 21 days, the percentage of survival was (100%)
and (95%) and virus free plantlets was (28.5%) and (42.8%) for C. morifolium
plants, respectively. These results were in agreement with (Ram
et al., 2005) whom stated that heat treatment (thermotherapy) at
elevated temperatures (34-42°C) was used to produce healthy plants from
individuals suffering from diseases such as potato witches broom and aster
yellows caused by phytoplasma.
Walkey (1991) reported that for obtaining virus-free
materials, addition of antiviral compounds were recommended in the absence of
heat treatment and meristem culture to eliminate viruses especially in the commercial
laboratories. Several investigators suggested that the contact of the virus-infected
tissues with the culture medium might eliminate viruses present in the tissue.
Moreover, the multiplication of viruses was usually decreased when infected
plants were treated with chemical analysis to the purine and pyrimidine bases
of nucleic acids. These analogues act like heat therapy to increase the success
of the apical meristem and tip culture technique.
Harris and Robins (1980), Kim et
al. (1994) and Ram et al. (2005) stated
that ribavirin (Virazole or 1- B-D-ribofuranosyl-1, 2, 4-triazole -3-carboxamide),
a base nucleotide analogues that has been extensively tested against human viruses,
appears to be effective against both RNA and DNA viruses. Many investigators
demonstrated that incorporation of ribavirin into culture media resulted in
an increased percentage of virus-free progeny.
The obtained results indicated that incorporation of ribavirin (virazole) in
culture medium at concentration of 10, 20 and 30 mg L-1 progressively
increase the percentage of virus-free plantlets to (29.4, 31.3 and 58%) in Chrysanthemum
plantlets respectively. Virazole proved to be somewhat phytotoxic to Chrysanthemum
plantlets but this effect disappeared gradually 15 days after culture and the
plantlets reversed to green color. The percentages of survival by using virazole
were (100, 93 and 86%) for Chrysanthemum plantlets, while Albouy
et al. (1988) reported that in Orchid cultures, two viral diseases
caused by Odontoglossum Ring Spot Virus (ORSV) and Cymbidium Mosaic Virus (CMV)
have been reported to limit the production. Attention of virus free orchids
by meristem tip culture has proved to be often inadequate. Different chemicals
such as virazole have been reported as means of freeing plant culture in
vitro from viruses. In order to increase the efficiency of eradication of
ORSV and CyMV, chemotherapy based on the incorporation of virazole in the solid
medium culture has been investigated on Cymbidium protocorms cultured in vitro.
Factors such as growth regulators and active charcoal are of critical importance.
Best results were obtained in shortening the time of transplantation (18 days)
allowing a good excision of Cymbidium new protocorms. After 5 subcultures of
18 days in presence of 25 ppm of virazole we obtained 95% virus free plantlets.
The obtained results concerning the combination between chemotherapy and thermotherapy by subjecting the infected shoots to 3^°C for 2 weeks produced (95, 90 and 85%) survival for Chrysanthemum while the percentages of virus free were (38, 61.5 and 76.8%) for 10, 20 and 30 mg L-1 virazole, respectively, showed the effects of thermotherapy and chemotherapy (virazole) on TMV elimination from infected Chrysanthemum plants.
Finally, the obtained results under this study concerning the combination treatments are promising to be an effective method for TMV eradication from Chrysanthemum plants in order to produce TMV-free Chrysanthemum plantlets via tissue culture technique.