Abstract: The present study was taken up to evaluate the effect of leaf extracts and acetone-precipitated protein of medicinal plants on seed-borne Tobacco Mosaic Virus (TMV) and Tomato Mosaic Virus (ToMV) infection. The antiviral activity was tested on indicator plant Nicotiana glutinosa. Acetone precipitated proteins and solvent extracts of six medicinal plants were tested for their effect on tobamovirus infection. The aqueous leaf extracts of Guava, Phyllanthus and Thuja were effective in reducing the infection by ToMV. The acetone-precipitated fractions of Tridax, Thuja, Guava and Tulsi were effective in reducing the infection by TMV. The solvent extract of Guava was effective in reducing the ToMV infection. Guava extract was subjected to TLC and the fractions were tested for their antiviral activity. Fraction with RF value of 0.014 proved to be effective in reducing the ToMV infection. The solvent extract of Thuja was effective in reducing TMV infection. TLC fraction of Thuja extract with the RF value of 0.12 reduced the TMV infection. Along with this, effect of aqueous leaf extracts on seed quality parameters of tomato and bell pepper was studied.
INTRODUCTION
Tomato (Lycopersicon esculentum L.) and bell pepper (Capsicum annuum L.) are the two important members of family Solanaceae, which are used as vegetables worldwide. Tomato Mosaic Tobamovirus (ToMV) and Tobacco Mosaic Tobamovirus (TMV) are among the important viruses that infect these crops. Yield loss in tomato due to ToMV infection has been reported to be 3 to 95% and in bell pepper, the loss due to TMV infection is between 1 to 90% (Chitra et al., 2002). Chitra et al. (1999) reported the seed transmission rate of ToMV to be 1-13% and that of TMV to be 1-10%. Control of viral infection by the application of antiviral chemicals has not been successful (Verma et al., 1984). Several plant species have been screened as sources of antiviral factors. (McKeen, 1956; Verma et al., 1984; Manickam and Rajappan, 1999; He and Liu, 2004).
The botanicals may induce resistance or they themselves may act as inhibitors of viral replication. Ribosome Inactivating Proteins (RIPs) and glycoproteins may block the replication sites. A mobile inducing signal may be produced in treated leaves after the botanical resistance inducers bind with the host plant surface. This signal produces virus-inhibiting agent in the entire plant system. Certain low molecular weight pathogenesis related proteins might also play a role in the induction of systemic acquired resistance (Verma et al., 1998).
Thus, biologically active compounds present in plant products act as elicitors and induce resistance in host plants resulting in reduction of disease development (Verma et al., 1998). The present work was carried out with an objective to screen promising botanicals and their fractions against seed-borne tobamoviruses. In this study, six botanicals viz. Psidium guajava (Myrtaceae), Leucas aspera (Lamiaceae), Ocimum sanctum (Lamiaceae), Tridax procumbens (Asteraceae), Phyllanthus niruri (Euphorbiaceae) and Thuja occidentalis (Cupressaceae) were screened for their antiviral activity against TMV and ToMV.
MATERIALS AND METHODS
Maintenance of Viruses, Host Plants and Indicator Plants
Seeds of Tomato PKM-1 (Ashoka Farm Aids) and Capsicum CW (Sultan Seed Farm)
were obtained from local seed agencies. Nicotiana glutinosa seedlings
(two month old) were maintained in an insect-proof screen house condition. TMV
was obtained from infected bell pepper plants and ToMV was obtained from infected
tomato plants maintained in the insect-proof screen house.
Preparation of Aqueous Plant Extracts and Acetone Precipitation of Proteins
Aqueous extracts were obtained by grinding 100 g fresh leaf of the plant
species with 100 mL of phosphate buffer (0.01M, pH 7.2) (1:1 w/v) by using pestle
and mortar. The extract was filtered through double-layered cheese cloth. The
filtrate was sonicated and centrifuged at the rate of 10,000 rpm for 10 min.
The supernatant was used for further studies. Proteins were precipitated from
the aqueous extracts by using cold acetone. Four volumes of cold acetone was
added to the supernatant. It was kept at 4°C overnight and centrifuged at
10,000 rpm for 15 min. The pellet was washed with acetone, centrifuged and dissolved
in minimum amount of phosphate buffer (0.01M, pH 7.2). Protein concentration
was estimated by the dye-binding method (Bradford, 1976).
Preparation of Solvent Extracts
Solvent extracts were obtained by grinding 10 g of the leaves in 10 mL of
95:5. Chloroform: Methanol (1:1 w/v). The extract was filtered through double-layered
cheese cloth. The solvent was evaporated and the residue was dissolved in a
known volume of methanol. This was used for further studies.
Isolation of Bioactives
Silica plates were prepared by preparing slurry of silica powder in water
in the ratio 1:2 (w/v). This slurry was poured onto the plates with the help
of an applicator. The slurry was coated onto the glass plates. The plates were
allowed to dry for 15-30 min. These plates were then heated in an oven at 100-120°C
for 1-2 h to remove the moisture and to activate the adsorbent (silica gel)
on the plate (Sadasivam and Manickam, 1991).
Fractions were collected from the crude solvent extracts that were effective in reducing the virus concentration on the indicator plants of N. glutinosa. The solvent extracts were passed through a silica column (60-100 mesh) and 4 mL fractions were collected. The fractions were spotted onto a silica plate of 0.3 mm thickness. Presence of a single UV fluorescent band confirmed the purity of the fraction. Retardation factor (RF) of each fraction was calculated using:
Treatment of the Plants
Precipitated Protein
Equal amount of protein (0.5 mg) was sprayed onto the leaves of N. glutinosa.
Challenge inoculation with TMV or ToMV was done after 1 h.
Crude Extract
The methanol extract was sprayed onto the leaves of N. glutinosa.
After one hour, challenge inoculation with the viruses was done. A control was
maintained.
The silica gel fractions were tested for their antiviral activity. The fractions were dissolved in methanol and sprayed onto the leaves of N. glutinosa. After one hour, challenge inoculation with the viruses was done.
Challenge Inoculation of Viruses
The leaves of tomato and bell pepper infected with ToMV and TMV were ground
well in pre-chilled mortar and pestle by using 10 mM Phosphate buffer (pH 7.2)
along with carborandum powder (240-1000 mesh) as abrasive. The N. glutinosa
leaves treated with the acetone precipitated proteins and solvent extracts
of the plants were inoculated with the viruses by using a cotton swab dipped
in the inoculum. The inoculum was then swabbed onto the leaves of the treated
plants. The leaves were washed with water after 10 min of inoculation of viruses.
The leaves were inoculated and after three days the inoculated leaves were observed for the formation of necrotic local lesions. The number of local lesions on inoculated leaf/100 cm2 was calculated using the formula:
Effect of Aqueous Leaf Extracts on Seed Quality
Four hundred seeds of tomato and bell pepper were placed in conical flasks
containing the plant leaf extracts (10% w/v). The flasks were kept on a rotary
shaker for 24 h. Distilled water was used as control.
The treated seeds were subjected to germination test using the between paper method (ISTA, 2003). Vigour index was calculated using the formula.
VI = (MSL + MRL) x% germination |
Statistical Analysis
The data generated was average of three independent experiments having three
replicates each. Data was subjected to Analysis of Variance (ANOVA) and the
means were compared for significance using Duncan’s Multiple Range Test
(DMRT; p = 0.05).
RESULTS
Effect of Acetone Precipitate of Botanicals on Tobamoviruses
The antiviral activity was assessed based on the number of local lesions
formed on control and treated N. glutinosa leaves. The leaves treated
with acetone precipitate of Guava (100), Tridax (49) and Thuja
(39) showed reduced number of local lesions due to TMV infection when compared
to control (385) and other acetone precipitated fractions (Table
1). Guava and Thuja acetone precipitate also reduced the number of
local lesions by ToMV. Tulsi fraction was also effective against ToMV.
| Table 1: | Antiviral activity of acetone precipitated proteins against TMV and ToMV |
| Every value represents the mean of three experiments with Standard Error (SE). The values with different letter are significantly different according to DMRT (p = 0.05) | |
| Table 2: | Antiviral activity of solvent extract (crude) against TMV and ToMV. |
| Every value represents the mean of three experiments with Standard Error (SE). The values with different letter are significantly different according to DMRT (p = 0.05) | |
| Table 3: | Antiviral activity of the silica gel fractions from Thuja leaves against TMV |
| Every value represents the mean of three experiments with standard error (SE). The values with different letter are significantly different according to DMRT (p = 0.05) | |
Effect of Solvent Extracts on Tobamoviruses
The solvent extracts were less effective in inhibiting the viruses. Only
Thuja extract was effective in inhibiting TMV. The number of local lesions
formed on N. glutinosa leaves treated with Thuja extract was 92
as compared to 193 in control (Table 2). Guava leaf extract
was found to be effective in reducing the ToMV infection. The number of local
lesions reduced to 66 in comparison to 100 in control (Table 2).
The fractions eluted from the silica gel column from the extracts of Thuja and Guava leaf extract were separated on TLC plates. The fractions having different RF value were used in the study. In all the fractions, a single band was seen under UV, which confirmed the purity of the fractions.
The fractions of Thuja leaf extract having RF value of 0.130, 0.136, 0.170, 0.178 and 0.205 showed reduced the number of local lesions to 40 to 62 in comparison to control (200) upon challenge inoculation with TMV (Table 3).
The fractions of Guava leaf extract having RF value of 0.046, 0.120 and 0.420 showed reduced the number of local lesions to 115, 63 and 77 when compared to control (364) upon challenge inoculation with ToMV (Table 4).
| Table 4: | Antiviral activity of the silica gel fractions from guava leaves against ToMV |
| Every value represents the mean of three experiments with Standard Error (SE). The values with different letter are significantly different according to DMRT (p = 0.05) | |
| Table 5: | Effect of aqueous leaf extracts on seed quality |
| Every value represents the mean of three experiments with Standard Error (SE). The values with different letter are significantly different according to DMRT (p = 0.05) | |
Effect of Aqueous Leaf Extracts on Seed Quality
The seeds of tomato and bell pepper treated with aqueous leaf extracts had
influenced the seed quality parameters. In tomato seeds treated with botanicals,
the maximum germination was found when seeds were treated with the aqueous extract
of Tulsi (93%) in comparison with control (85%) and other treatments. Seeds
treated with aqueous extracts of Leucas and Tridax showed 84 and
82% germination respectively. But seed treatment with Guava leaf extract reduced
germination to 56%. In tomato seeds treated with Tulsi, vigour index increased
to 1166 whereas in Guava, it reduced to 748 from 1122. Maximum mean shoot length
was observed in tomato seeds treated with Tridax (7.51) along with Leucas
(7.25) in comparison to control (6.91) and other treatments. Tomato seeds treated
with Guava (6.61), Thuja (6.43) and Tulsi (6.49) leaf extracts showed
increased mean root length when compared to control (6.29) and other treatments
(Table 5).
In bell pepper seeds treated with botanicals, increase in germination was noticed in seeds treated with extract of Phyllanthus (56%) in comparison to control (54%) and other treatments. Seed treatment with Leucas and Thuja extracts showed 53 and 52% germination, respectively. But seed treatment with Tulsi extract reduced germination 54%. Vigour was more in seeds treated with Leucas (421) and Phyllanthus (534) extracts when compared to control (392). In seeds treated with Tulsi extract, vigour reduced to 131. Maximum mean shoot length was observed in seeds treated with extract of Phyllanthus (5.19) in comparison to control (3.02) and other treatments. The mean shoot length increased in seeds treated with extracts of Leucas (3.86), Tridax (3.10) and Thuja (3.32). The mean shoot length was minimum in seeds treated with Tulsi extract (2.24) when compared with control and other seed treatments. The mean root length was maximum in seeds treated with extract of Phyllanthus (4.30) when compared to control (3.02) and other treatments. Seeds treated with Leucas extract showed a mean root length of 4.24. The mean root length was minimum (2.87) when seeds were treated with Guava leaf extract. The mean root length decreased in seeds treated with Tridax (3.72), Thuja (4.01) and Tulsi (2.95) extracts in comparison to control (Table 5).
DISCUSSION
In the present study, an attempt was made to control TMV and ToMV by using certain plant extracts. The effect of these extracts on germination and vigour was also evaluated. Plant extracts have gained importance in the recent days because of their safety and target specificity. The plant constituents are easily bio-degradable, less phytotoxic and more systemic. Six different plant species viz, Psidium guajava, Leucas aspera, Phyllanthus niruri, Tridax procumbens, Thuja occidentalis and Ocimum sanctum were evaluated for their antiviral activity against ToMV and TMV. In a similar way, many workers have reported the use of some of these plants for the control of viruses (Mandal and Sigh, 2001; Jayashree et al., 1999).
Mandal and Sigh (2001) reported that Turnip Mosaic Virus was effectively inhibited by guava leaf extract. In our experiments also, the acetone precipitated fraction of guava inhibited both TMV and ToMV whereas the solvent fraction inhibited ToMV. Jayashree et al. (1999) reported that a plant derivative from Thuja, called Thuja 30 inhibited the pumpkin yellow vein mosaic virus. In our experiments, the solvent extract of Thuja inhibited only TMV whereas the acetone-precipitated fraction inhibited both TMV and To MV.
In our experiments, four of the six plants showed antiviral activity. The acetone-precipitated fractions of Guava, Thuja and Tulsi inhibited only TMV. Leucas and Phyllanthus did not show any inhibitory activity. Baranwal and Verma (1997) have reported the inhibition of TMV by leaf extract of C. cristata. Madhusudhan et al. (2005) have reported the antiviral activity of neem oil. At a concentration of 5%, neem oil reduced the concentration of TMV and ToMV. The leaf extract of Celosia cristata was found to prevent lesion production by sunnhemp rosette virus, TMV and PVX in several hosts (Baranwal and Verma, 1992). Wang and Tumer (1999) and He and Liu (2004) confirmed the RNAse activities of highly purified pokeweed antiviral protein and a recombinant cinnamomin A chain from Cinnamomum camphora. McKeen (1956) showed the inhibition of cucumber mosaic virus infection in untreated opposite primary leaf of cowpea whose other primary leaf was treated with extracts of C. frutescens.
The untreated upper leaves of Cyamopsis tetragonoloba were disease-free whose basal leaves were treated with leaf extract of different species of Clerodendrum a day before virus inoculation (Verma et al., 1984). Green gram leaf curl disease, which is caused by Tomato Spotted Wilt Virus (TSWV) was effectively controlled by using plant extracts and chemicals. Among 11 botanicals used, Cocos nucifera, Sorghum vulgare, Euphorbia thyifolia, Prosopis chilensis, Croton sparsiflorus were found to be effective in controlling Tomato spotted wilt virus when compared to control (Manickam et al., 1999). Almost all the virus inhibiting substances from higher plants are recognized as basic proteins. The roots, leaves and stem of Mirabilis jalapa show high inhibitory activity against plant viruses. Verma and Kumar (1980) showed that M. jalapa leaf extract suppressed the disease symptoms on a few systemic hosts when the extract was used as a foliar spray 24 h prior to virus inoculation. In our experiments, the leaf extracts of Guava, Tulsi, Thuja and Tridax reduced the concentration of the tobamoviruses effectively.
The antiviral activity was assayed by the number of lesions on the indicator leaf. The reduction in the number of lesions indicated the resistance of the plant to the virus. Verma et al. (1996) reported the antiviral activity of C. aculeatum against mechanically and white fly transmitted viruses by this method. Praveen et al. (2001) have reported the induction of resistance to be maximum when the inducer molecule was applied on plants before 40-60 min of virus inoculation. In our experiment, challenge inoculation with the virus was done 60 min after the treatment.
Methanol, chloroform and hexane extracts of Maesa lanceolata were found to effective against fungal plant pathogens (Okemo et al., 2004). In our experiment, Chloroform:Methanol (95:5) extracts of six test plants were screened against TMV and ToMV. Guava and Thuja crude extracts showed maximum reduction in local lesions formation in N. glutinosa challenge inoculated with ToMV and TMV respectively. The fractions of Thuja and Guava having RF value of 0.12 and 0.014 showed maximum reduction in local lesions formed with TMV and ToMV, respectively when compared to control leaf and other fractions.
The seeds of tomato and bell pepper were treated with the plant extracts for 24 h at 10% concentration. The germination percentage and vigour index were evaluated. In some extracts, the germination and vigour index reduced. This discrepancy might be due to the phytotoxicity of the extract on seed quality. Few extracts did not effect of the treatment on the seed germination and growth, which may be due to the non-penetration of the active principle through seed coat.
The results obtained by this research supports that use of botanicals can be useful strategy to reduce the incidence of viruses. The botanicals may induce resistance or they themselves may act as inhibitors of viral replication. Thus, biologically active compounds present in plant products act as elicitors and induce resistance in host plants resulting in reduction of disease development.
Based on the results obtained in our studies, acetone precipitated proteins from leaves of Guava, Tridax and Thuja were effective against TMV where as Tridax, Thuja, Guava and Tulsi were effective against ToMV. The silica gel fractions isolated from Thuja and Guava having RF value of 0.136 and 0.12 showed maximum reduction in local lesions formed on N. glutinosa challenge inoculated with TMV and ToMV, respectively.
The extracts of four plants and the silica gel fractions from two plants have proved effective in reducing the tobamovirus concentration on N. glutinosa leaves. Further studies like characterization of the metabolite, structure elucidation, formulations that can be used to treat the host plants etc. needs to be carried out.