Abstract: This is the first record of Tobacco mosaic Tobamovirus (TMV-S) on sunflower in Egypt. It was originally isolated from naturally infected sunflower plants growing in Giza Research Station, showing systemic mosaic and spots. Purified TMV-S migrated as a single zone in density gradient column. Ultraviolet absorbance of TMV-S was typical of nucleoprotein with minimum and maximum at 247 and 260 nm, respectively. The ratios of A260/280 and Amax/min were 1.2 and 1.1, respectively. Electron microscopy of purified virus showed the presence of rod shape particles with a size 300 nm. Titer of the prepared antisera as determined using ELISA was 1/2000. Electron microscopic examination of infected leaves of N. clevelandii found various cytological abnormalities. Due to the non-availability of sources of resistance in Egypt to TMV-S in sunflower, a mutation breeding program was initiated. Seeds of two genotypes were subjected to four doses of gamma rays; 0, 100, 200 and 300 Gy from a 60Co source. M1 and M2 generations were sown at the experimental farm of the Agricultural Research Center to induce variability in the sunflower genotypes; Giza 102 and Sakha 53 which could be resistant to TMV-S. The statistical analysis indicated significant differences among irradiation doses on plant height; all used doses increased plant height comparing with the control. Inoculation with TMV-S caused decreases in plant height over all other factors. Number of leaves differed significantly according to the used cultivars, gamma ray doses and TMV-S. The highest number of leaves were found during M2 generation. The differences in head diameter between cultivars were significant. The highest head diameter was observed in Giza 102 (21.7 cm) in M1 generation, followed by 20.2 and 20.0 cm for Giza 102 genotype developed through irradiation with 300 Gy in M2 generation and Sakha 53 genotype developed through irradiation with 300 Gy in M1 generation. Seed yield per plant differed significantly between all cultivars, irradiation doses and infection statuses. The 100 Gy of gamma ray irradiation doses decreased seed yield, while 200 and 300 Gy gamma ray dose increased seed yield per plant for both cultivars in both generations, the highest seed yield per plant were observed with Sakha 300 Gy in both generations, it was 104.1 g (M2) and 102.1 g (M1), while the highest seed index (100-seed weight) was noticed for Giza 102 genotype developed through irradiation with 200Gy in M1 generation. It seems that 200 and 300 Gy treatments increased the most of studied characters in M1 and M2 generations in sunflower. The increments in mean of head diameter as a result of applying 300 Gy gamma ray dose increased seed yield/plant and seed index which finally improved seed yield. Both morphological characters and seed yield were reduced significantly as a result of virus infection. The effect of treatment with the virus was negative in all irradiated Sakha genotype developed through irradiation with 100, 200 and 300 Gy, as well as and Giza 102 genotype developed through irradiation with 100 Gy, because these genotypes tend to be resistant to TMV-S as ELISA test reported.
INTRODUCTION
Sunflower (Helianthus annuus) is the most important source of edible oil in Zambia. It is widely grown and it is an important cash crop. Virus-like symptoms characterized by yellow blotching of leaves have been observed, especially among the newly introduced sunflower cultivars since 1988. Severe stunting of plants and almost 100% disease incidences has been recorded on some farms. Preliminary studies have indicated that T. procumbens is an alternate host to this viral disease and that aphids are capable of transmitting the disease from this host to sunflower (Muunga, pers. comm.). The disease is thought to be caused by Sunflower Yellow Ring Spot Virus (SYRSV) or Sunflower Yellow Blotch Virus (SuYBV) genus Umbravirus reportedly infecting groundnut. A similar virus disease is reported to occur on sunflower in Kenya (Theuri et al., 1987). Although, several new viruses belonging to the genus Tobamovirus have been described (Lewandowski, 2005). Tobamoviruses are known as serious plant pathogens, in particular the type species, Tobacco mosaic virus (TMV-S), is considered one of the most dangerous plant viruses. Tobamoviruses are easily transmitted mechanically, without the help of vectors. Geographical distribution of Tobamoviruses is world wide (Lewandowski, 2005). There is still an urgent need to fully characterize this disease in order to formulate appropriate control strategies against it. This paper was initiated to: (1) Detection of virus (TMV-S) in sunflower plants by electron microscopy. (2) Production of antiserum specific to TMV-S (3) yield losses.
MATERIALS AND METHODS
Virus source and symptoms: Samples of sunflower (L). G. Don. plants showing typical systemic mosaic and yellowing symptoms of TMV-S were collected from Agriculture Research Experimental Station (ARES).
Isolation and propagation: The virus was isolated by repeated single lesion isolation on leaves of Chenopodium amaranticolor L. and then propagated in Nicotiana tabacum L. cv Samsun as described previously by Da Silva et al. (2008).
Purification of TMV-S: The method used in this study is similar to that described by Takahashi and Ahohara (1990) with minor modification. Rate zonal centrifugations in the sucrose density-gradients were made instead of cesium chloride (CsCl) solutions. Beside that, the final pellet was resuspended in potassium phosphate buffer, pH 7.2 instead of citric acid buffer, pH 8.2 extraction buffer. Chloroform only was used instead of carbon tetrachloride and ethyl-ether. TMV-S infected sunflower leaves, propagated by sap-inoculation, were designated as a testing material. Purification was performed by putting 0.1 M sodium citrate buffer (pH 6.8, 3X w/v) in the infected leaves, grinding them for 30 min in a 4°C cold room and then filtrating through cheese cloth. After stirring up 30% Chloroform for 10 min, several centrifugation steps were followed. The supernatant was centrifuged through a 10-40% linear sucrose gradient in 50% potassium phosphate buffer, pH 7.5 at 32,000 rpm for 2.5 h in a Bekman SW 60 rotor. Gradient columns were made and stored overnight at 4°C prior to use. The virus zones were collected with a bent tip hypodermal needle and syringe. The virus zone was diluted 1:1 with 0.05 M potassium phosphate buffer, pH 7.5 and the concentrated by High Speed Centrifugation (HSC). The resulting pellets were resuspended in 2 mL of the buffer and stirring overnight at 4°C. The purified virus preparation was estimated spectrophotometrically to evaluate the purity and concentration, using an extension coefficient E0.1% 2.2 for TMV-S (Converse and Martin, 1990) with a spectronic 2000 spectrophotometer. The OD260 was converted to mg of virus/mL. Infectivity was tested on leaves of C. quinoa.
Electron microscope examination: Purified virus preparation was negatively stained with 2% phosphotungstic acid (FTA), pH 7.0 and examined with an electron microscope (Noordam, 1973; Kim and Lee, 1999). A drop of virus preparation was placed on carbon coated grid for 2 min. After filtration, a drop of 2% phosphotungstic acid (FTA) was added. After 2 min more the excess liquid was drained. The treated grid was examined using JEOL-JEM-1010), Faculty of Science, Al-Azhar University.
Antiserum and ELISA
Reagents production
Production of antiserum specific to TMV-S
Rabbit immunization: New Zealand white rabbits, about 3 kg weight was
used for antiserum raised against TMV-S. A total of 8.5 mg purified TMV-S were
used for subcutaneous. Purified virus preparation was emulsified with an equal
volume of Freud's complete adjuvant for the first and incomplete adjuvant for
six subsequent injections at weekly intervals. Subcutaneous injections were
made into the neck area of the rabbit by pulling up the loose skin and inserting
a 22-gauge needle between the skin and muscle tissue (Hampton
et al., 1990; Da Silva et al., 2008).
Rabbit bleeding and blood collection: Rabbits were bled one week after the last injection along five weeks, from the right ear. The blood was collected, left to clot at 37°C in an incubator for 1-2 h and then kept at 4°C overnight. Antiserum was separated through centrifugation at 5,000 rpm for 3 min and stored at -20°C dispended as small aliquots in coated tubes until used for titer determination and other serological tests.
Determination of antiserum titer: Checkerboard titration was used to determine optimal conditions for indirect micro plate ELISA for TMV-S antisera.
Clarified sap of virus infected and control squash leaves were diluted at 1/5, using phosphate buffer, pH 7.5, containing 0.85% NaCl. TMV-S antiserum preparations were diluted with the serum buffer, 1/500. 1/1000, 1/500, 1/1000, 1/1500, 1/2000, 1/2500, 1/3000, 1/3500 and 1/4000, respectively. The reaction was done between infected clarified extract and its induced antiserum by indirect ELISA test (Converse and Martin, 1990).
ELISA reagents production
Purification of the immunogamaglobulin G (IgG): Gamaglubulins were purified
from the antisera using the caprylic acid method recorded by Steinbuch
and Audran (1969):
| • | One milliliter of virus antiserum was added to 0.06 M sodium acetate buffer, pH 4.8 (1:2) |
| • | IgG was dialyzed against this buffer (0.06 M sodium acetate) for about 24 h (three times) at 4°C. |
| • | While stirring vigorously drop-wise 0.082 mL caprylic acid were added with continuous stirring for 30 min at room temperature, then centrifuged at 8,000 rpm for 10 min |
| • | The supernatant was collected and dialyzed twice against 0.05 M phosphate buffer, pH 7.2, for 4 h at 4°C. |
| • | The resulting IgG was diluted with distilled water to make 4 mL and an equal volume of saturated ammonium sulphate solution were added at room temperature while stirring and the stirring was continued for 30 min. |
| • | After centrifugation at 8,000 rpm for 10 min, the pellet was collected and suspended in 1 mL distilled water followed by dialysis three times against 0.05 M phosphate buffer, pH 7.2, for 24-48 h at 4°C. If necessary the IgG was centrifuged clear 10 min at 8,000 rpm. The IgG was then adjusted to 1 mg mL-1 (A280 nm = 1.4) and stored at -20°C in coated tubes until use |
Conjugation of IgG with alkaline phosphatase:
| • | The bottle of alkaline phosphatase (0.7 mg AP Sigma P-5521, 2,000 units) was washed with 3.2 M ammonium sulfate, pH 7.0 |
| • | The precipitate was collected after centrifugation 20 min at 6.000 rpm and dissolved in 0.350 mL IgG (1 mg mL-1) at the rate of 2 mg AP/1 mL IgG |
| • | The conjugate (IgG-AP) was dialyzed 5 times in PBS buffer, pH 7.5. The last dialysis, PBS buffer without NaN3 was used |
| • | Glutaraldehyde (1%) was added to a concentration of 0.06% (VIgG-APx0.06 = Vg) and the mixture was incubated 4 h at room temperature with gentle stirring. A light yellow-born color was developed |
| • | Glutaraldehyde was then removed by dialysis 5 times in PBS buffer with NaN3 (0.02%), the volume of the conjugate was measured and BSA (bovine serum albumin, Sigma A-4503) at the rate of 5 mg BSA to 1 mL solution was added |
| • | The conjugate either dispended as small aliquots or diluted with glycerol to 50% (v/v) and stored at -20°C in coated tubes, where it should remain stable for many months. Because of volume changes and possible gama-globulin losses during the conjugation procedure all references to the use of conjugates are in terms of dilutions of the conjugate rather than at absolute concentrations |
Electron microscopy: Sections of leaves exhibiting acute symptoms was prepared for electron microscopic examination. Leaf specimens were cut 1-2 cm long then fixed in 2.5% glutaraldehyde in potassium phosphate buffer, pH 7.4. Specimens were washed in cold buffer and postfixed in 1% osmium tetroxide in the same buffer for 3 h. Sections of leaves from healthy control plants were similarly prepared for electron microscopy. After staining overnight in 1% uranyl acetate (Gardner, 1967), the leaf specimens were dehydrated in ethanol-acetone series and embedded in Spurr’s medium (Spurr, 1969). Ultra-thin sections were cut with glass knife on LKB ultramicrotome, mounted on copper grids and stained for 10 min with a mixture of an equal volume of saturated uranyl acetate and acetone followed by lead citrate (Kim and Lee, 1999; Da Silva et al., 2008).
Irradiation and field experiments: Dryseeds(10%moisture content) of two sunflower cultivars: Giza 102 and Sakha 53 (obtained from the Oil Crops Research Department, Field Crops Research Institute, ARC) were exposed to 0, 100, 200 and 300 Gy of gamma rays at a dose rate of 0.8 Gy min-1 at the National Center for Radiation Research and Technology, Atomic Energy Authority. Irradiated seed lots and non-irradiated controls were grown (immediately after irradiation) on 15th of June, 2009 at Giza Research Station, ARC to give M1 generation. At harvest, data were recorded on morphological and yield components characters. Seeds of each genotype were bulked separately.
In M2 generation, seeds from each irradiated M1 treatment as well as controls were grown on 21st of June, 2010 to obtain M2 plants.
At harvest, data were recorded on: In M1 and M2 generations, a split-split plot design with four replications was used for each generation. The two infection statuses were devoted to main plots, two cultivars to subplots and four irradiated doses to sub-sub plots. The irradiated and non-irradiated seeds were sown in plots; each plot consisted of five rows, 4 meters long, 70 cm apart and 20 cm between hills. Random samples of 20 individual plants for both un-infected (healthy) and infected with TMV-S per treatment were used to measure studied characters: plant height (cm), number of leaves, head diameter (cm), seed yield/plant (g) and 100-seed weight (seed index) (g).
All data were statistically analyzed by the software CoStat pro (2005) in consultation with the analysis of variance (Gomez and Gomez, 1984). The means were compared using Least Significant Difference (LSD) at p = 0.05 as outlined by Duncan (1995).
RESULTS AND DISCUSSION
In Egypt, This first reported the occurrence of a new Tobamovirus infect cultivars sunflower. The virus was serologically with TMV-S even though it shared the same antigenic sites. Then the Tobacco mosaic virus sunflower strain (TMV-S-S) has been used for the new virus Tobamovirus. Do not learn to know precisely an isolate TMV-S which effect sunflower approach to any strains for TMV-S, after an analysis Nucleotide sequence special for the virus and this will be later.
Isolation and propagation: The virus isolate, TMV-S was isolated from infected sunflower. After biological purification through single lesion transfers on C. amaranticolor, the resulting virus was propagated on either N. rustica on Helianthus annuus.
Transmission studies
Mechanical transmission: TMV-S was easily transmitted mechanically
to sunflower inoculated plants with showed chlorotic local lesions followed
by systemic chlorosis (Fig. 1). It causes systemic chlorosis
on seed heads (Fig. 2).
Virus purification and ELISA kit production
Purification of the virus isolate: Purified TMV-S migrated as a single
zone, 3 to 4 cm, respectively below the meniscus of the density gradient column.
This zone was found infections when tested in the local lesion host plant and
gave typical ultraviolet absorption spectrum of nucleoprotein with a maximum
and a minimum at 260 and 247, respectively (Fig. 3).
A260/280 and Amax/min ratios were 1.2 and 1.1 for TMV-S. These results are in agreement with other investigators (Brunt et al., 1996).
| Fig. 1: | Chlorotic local lesions followed by systemic chlorosis on sunflower inoculated with the virus isolate |
| Fig. 2: | Symptoms shown include systemic chlorosis and reduction in size of seed heads |
| Fig. 3: | Ultraviolet absorption spectra of purified TMV-S |
Electron microscopy: Extracted virus particles from infected sunflower leaves were observed by an electron microscope using a dip method. Typical rod shape virus particles, 300 nm long were found (Fig. 4). Such results agree with the diameter values reported for virions of TMV-S by (Kim and Lee, 1999).
Serologic studies:
Production of antiserum specific to TMV-S: Polyclonol antibodies
raised against TMV-S was prepared. The antisera produced against TMV-S had titers
of 1/2000 as determined by indirect ELISA test (Table 1).
In the present work, the polyclonal antibody raised against TMV-S had a virus-specific
titer of 1:2000 which was successfully used in ELISA technique. The concentration
of IgG after purification and the IgG conjugated with alkaline phosphatase was
1:1000.
One of the major goals in the present work is to produce ELISA reagents which can be used as a rapid method for TMV-S detection (Converse and Martin, 1990).
Cytological studies:
Electron microscopy: Electron microscopic examination of infected
leaves of N. clevelandii revealed various cytological abnormalities which
have been absent in healthy tissues. These abnormalities have been absent in
healthy tissues. These abnormalities are.
Ultra thin section of TMV-S infected N. clevelandii leaf showing nucleus which is misshapen and the chromatines are degenerated (Fig. 5, 6). Cytological studies revealed abnormalities in nucleus described for many other Tobamoviruses (Francki et al., 1985). Sabanadzovic et al. (2008) showed that virus particles were plentiful, forming large layered aggregates in the cytoplasm of infected petunias by TMV.
They often associated with plasmalemma and other membranes. The grana and intergrana appeared abnormal (Fig. 7) nuclei were notably abnormal, usually appear as more or less ellipsoidal bodies, they contained intranuclear inclusion of various sorts that may affect the nucleolus or the size and shape of the nucleus. Sometimes the nuclear membrane have became obscure and broken and also showed virus like particles in the cytoplasm (Fig. 8).
Previous identification of intracellular TMV by electron microscopy has been mentioned by Thomas (1964) extension of distorted encircling a mitochondria and containing a large and numerous TMV particles.
| Table 1: | Absorbance values of TMV-S antiserum |
| Fig. 4: | Particles of purified TMV-S preparation obtained from infected sunflower. The scale bar represents 0.5 m |
| Fig. 5: | Electron micrograph of ultra thin section of TMV-S infected N. clevelandii leaf showing nucleus which is misshapen and the chromatines are degenerated (x12,000) |
| Fig. 6: | Magnified part of fig 4 the misshapen nucleuos and the mitochondrion which begon in degeneration (x20,000) |
| Fig. 7: | An electron micrograph of swollen chloroplast (CL). Note that the grana and intergrana lamella were degenerated (x20,000) |
| Fig. 8: | Electron micrograph of mesophyll cell of TMV-S infected N. clevelandii showing virus like particles in the cytoplasm (x20,000) |
They are typical for many Tobamoviruses. Ladipo1 et al. (2003) observed the same cytopathological changes in infected cells of N. benthamiana, massive cytoplasmic inclusions consisting of stacked plate-like layers of virus particles. Each layer was a lateral aggregate of the rod-like particles with the particle ends in register. Presumably, due to fixation artifacts, the plate-like layers were also found in disordered arrangements. Also Dae et al. (2005) mentioned that cells infected with TMV-S in the pepper cultivars of Cheongyang and Wangshilgun had the typical ultrastructures of tobamovirus as the stacked-band structure and multiple Spiral Aggregate (SA). The cells infected with TMV-S had large numbers of Tobamovirus particles and accumulated in the cytoplasm and vacuole. TMV-S had the typical ultrastructures of Tobamovirus as the stacked and structure in cytoplasm. Electron microscopic examination will contribute towards a rapid and clear identification of virus diseases of plants and will be useful for diagnostic purposes in agriculture and in plant phytopathology (Bernd and Gunther, 2009).
Effect of Gamma ray irradiation treatments on sunflower characters in M2 generation: The mean values of studied characters for two sunflower cultivars in M1 and M2 generation after irradiation with gamma ray doses are presented in Table 2.
Significant differences were found between the two sunflower cultivars, gamma ray doses and treatment with TMV-S for plant height, number of leaves, head diameter, seed yield y. In this respect, A land seed index in both M1 and M2 generations. The highest genotype was Sakha 53 (Table 2). The highest plant height was observed in Sakha 53 treated with 200 Gy in M2 generation (187.0 cm), followed by Giza 102 treated with 300 Gy (183.0 cm). The statistical analysis indicated significant differences among irradiation doses on plant height; all used doses increased plant height comparing with the control. Inoculation with TMV-S caused decreases in plant height over all other factors.
Number of leaves differed significantly according to the used cultivars, gamma ray doses and TMV-S. The highest number of leaves were found during M2 generation, it was 36.7 (for Sakha 53 genotype developed through irradiation with 200 Gy) followed by 35.7 (for Giza 102 genotype developed through irradiation with 300 Garge spectrum of variability for morphological characters was isolated and characterized by Jambhulkar and Joshua (1999), among them 3 were for chlorophyll, 9 for leaf, 3 for stem and 8 for capitulum.
| Table 2: | Effect of TMV-S infection on mean values of morphological, yield and yield component characters for two irradiated sunflower cultivars in M1 and M2 generations in two successive seasons 2009 and 2010 |
| +: Positive, -: Negative | |
All of them bred true in subsequent generations. Distinct mutants were yellow leaf vein, fasciation and zigzag stem. Three characters were mutated in the wrinkled leaf mutant: the lamina was dark green and highly wrinkled, the petioles were thick and shortened and the ray florets were dissected. These novel mutations have not been reported so far, among the large number of mutations for morphological characters that were isolated and characterized for their inheritance pattern earlier (Luczkiewicz, 1975). Single recessive gene and two genes with complementary effect controlled most of them. Similar gene action for various morphological mutants has also been reported (Miller, 1992). Genetic analysis of the fascinated mutant in our studies showed that it is governed by a single recessive gene.
The differences in head diameter between cultivars were significant. The highest head diameter was observed in Giza 102 (21.7 cm) in M1 generation, followed by 20.2 and 20.0 cm for Giza 102 genotype developed through irradiation with 300 Gy in M2 generation and Sakha 53 genotype developed through irradiation with 300 Gy in M1 generation.
Seed yield per plant differed significantly between all cultivars, irradiation doses and infection statuses. 100 Gy of gamma ray irradiation doses decreased seed yield, while 200 and 300 Gy gamma ray dose increased seed yield per plant for both cultivars in both generations, The highest seed yield per plant were observed with Sakha 300 Gy in both generations, it was 104.1 g (M2) and 102.1 g (M1), while the highest seed index (100-seed weight) was noticed for Giza 102 genotype developed through irradiation with 200 Gy in M1 generation.
It seems that 200 and 300 Gy treatments increased the most of studied characters in M1 and M2 generations in sunflower. The increments in mean of head diameter as a result of applying 300 Gy gamma ray dose increased seed yield/plant and seed index which finally improved seed yield.
Both morphological characters and seed yield were reduced significantly as a result of virus infection. The effect of treatment with the virus was negative in all irradiated Sakha genotype developed through irradiation with 100, 200 and 300 Gy, as well as and Giza 102 genotype developed through irradiation with 100 Gy, because these genotypes tend to be resistant to TMV-S as ELISA test reported.