Antiviral activity of Thuja orientalis extracts against watermelon mosaic virus (WMV) on Citrullus lanatus

Abstract

Watermelon mosaic potyvirus (WMV) is considered as an important virus infecting watermelon and causing adverse effects on crop productivity. To overcome this problem one of the main objectives of plant breeders is to make these strains less effective in the ability to infect plants by treatment with plant extracts. Due to the advantages of plant tissue culture, in vitro, in the process of the selection of different cultivars under biotic stress, this study was conducted to achieve this aim by evaluating the effect of three concentrations of Thuja extract on the multiplication of WMV in watermelon by measuring callus fresh weight and soluble proteins (mg g⁻¹ fresh weight) of healthy and infected hypocotyl explants. Also, WMV was isolated from naturally infected watermelon and characterized as potyvirus by serological and molecular analyses. The isolated virus gave a positive reaction with WMV antiserum compared with other antibodies of CMV, ZYMV and SqMV using DAS-ELISA. RT-PCR, with the specific primer for WMV-cp. gene, yielded 825 base pair DNA fragments. The results that belong to soluble protein analysis indicated that infected hypocotyl explants treated with 6 g L⁻¹ recorded the highest rate in the number of soluble protein bands compared with the rest of treatments. As a conclusion of these results, we can recommend to apply the Thuja extract at 6 g L⁻¹ as a optimum dosage to decrease the infection caused by watermelon mosaic potyvirus.

1. Introduction

Numerous viruses have an effect on cucurbits and source of mosaic diseases Ali et al., 2012. The most important of these viruses are watermelon mosaic virus (WMV), cucumber mosaic virus (CMV), squash mosaic virus (SqMV), and tobacco ring spot virus (TRSV) Ali and Kobayahsi (2010). WMV is, one of the first potyviruses that is celebrated and one of the major significant potyviruses in the world (Aisan et al., 2012). This virus infects all tissues and causes adverse effect in cucurbits especially watermelon and that depends on the host plant and its age when infected. Watermelon plants are commonly stunted or dwarfed with yellow or light green mottling, leaf deformation, blistering, and marginal yellowing. Infected watermelon fruit by virus is often dwarfed, misshapen, mottled, or spotted (Aisan et al., 2012). WMV is composed of flexuous, filamentous particles, approximately 760 nm long and its genome is a positive-sense, single-stranded RNA about 10035nt (nucleotide) long (Al-Saleh et al., 2009; Zohreh, 2011). The first report of full sequenced WMV was from France (WMV-Fr) (Desbiez and Lecoq, 2004) followed by Chine (WMV-CHN) (Wu et al., 2006) and Pakistan (WMV-PK) (Ali et al., 2006).

Commercial cultivars of watermelon still lack effective resistance against the virus and the introduction of resistant genes into commercial cultivars by conventional breeding requires years of selection to eliminate unfavorable characteristics, even when a natural source is available (Compton and Gray, 1993b). Therefore, the treatment of plants with plant extracts and synthetic chemicals under in vitro culture will be the alternative method which can lead to the induction of resistant agents, that are characterized by restriction of virus multiplication and suppression of disease symptoms compared with untreated plants (Hammerschmidt, 1999; Al Ani and Hassan, 2002; Al-Ani et al., 2002, 2010; Walters et al., 2005). The main obstacle to the development of effective chemotherapy, is the nature of virus multiplication in the host cells (Yarmolinsky et al., 2009), In addition to that some viruses persist in a latent infection in the host (Horvath, 1983; Hull, 2002).

This study was to isolate the watermelon mosaic virus in fruits contracted from natural infection and identification was done by the ELISA test and RT-PCR, in addition, this study was also undertaken to evaluate the effect of different concentrations from Thuja orientalis extracts on virus multiplication through the determination virus concentration, callus fresh weight and total soluble proteins of healthy and infected hypocotyl explants.

2. Materials and methods

2.1. Watermelon mosaic virus sources and assessment of viral isolation

The original WMV isolate was maintained from natural infection in watermelon plants exhibiting green mosaic, blistering, vein banding and malformation were collected from different fields of the Jeddah city. Watermelon leaf samples were checked serologically by double antibody sandwich enzyme-linked immuno-sorbent assay DAS-ELISA technique according to the manufacturer’s instructions (Sanofi-Sante Animal, France) and were applied against WMV as described by Clark and Adams (1977). All samples were tested in triplicate using conventional (DAS-ELISA). Optical density was measured at λ = 405 nm in an ELISA micro well reader (using Dynatech Immunoassay MR 7000). Samples with an absorbency of at least twice that of the healthy controls were considered as positive for the presence of virus. Plant samples which gave a positive reaction in the direct ELISA test with WMV were separated and used as source of the virus. On the other hand, the DAS-ELISA technique was applied to measure WMV concentrations in all treatments including the healthy callus.

2.2. Total RNA extraction and RT-PCR

Total RNA was extracted from pulverized tissue using AccuZol™ Reagent (Bioneer, Alameda, CA) from fresh healthy and systemically infected watermelon leaves according to the manufacturers instructions. After precipitation with ethanol, total RNA was re-solubilized in 25 μL of RNase-free water. RNA samples were tested for the presence of WMV using specific primers designed to amplify a fragment of the coat protein gene. To amplify a 825-bp fragment covering the CP region, forwarded primer (5′-GAA TCA GTG TCT CTG CAA TCA GG-3′) and reverse primer (5′-ATT CAC GTC CCT TGC AGT GTG-3′) (Sharifi et al., 2008; Moradi and Jafarpour, 2010) corresponding to (GenBank accession number EU660584) nucleotides (nt) 8926–8948 and nt 9727–9747 of WMV-Fr, respectively. Reverse transcription (RT) reaction was performed as follows: 1 μL of reverse primer (20 pmol) and 1 μL of RNA sample were added to 8.5 μL of Diethyl Pyrocarbonate (DEPC)-treated water. The mixture was incubated at 65 °C for 10 min and chilled on ice for 3 min to denature the RNA. Then 3.5 μL of DEPC-treated water, 4 μL of 5× M-MLV RT buffer, 2 μL of dNTPs mix (10 mm) and 1 μL M-MLV (200U μL) reverse transcriptase (Promega, USA) were added to the mixture. The RT reaction was incubated at 42 °C for 60 min followed by 95 °C for 3 min to terminate the RT reaction. Viral cDNA was then amplified by PCR. The PCR reaction was performed using 2.5 μL of cDNA, 13.5 μL DEPC-treated water, 5 μL of 5× GoTag polymerase buffer, 2.5 μL 10× MgCl2, 0.5 μL of each forward and reverse primer (20 pmol), 0.75 μL of dNTP mix (10 mm) and 0.125 μL of GoTag polymerase (2.5 U μL) (Promega, USA). The following program was used for PCR. A first denaturation for 60 s at 94 °C, 60 s of annealing at 63 °C extension for 1 min at 72 °C and a final extension step at 72 °C for 10 min. PCR products were analyzed by electrophoresis in 1.7% agarose gel and visualized by ethidium bromide staining.

2.3. Efficiency evaluation of different concentration from Thuja extract on WMV infection

Thuja extract at 2, 4 and 6 g L⁻¹ were prepared according to Al-Ani et al. (2010). Samples of T. orientalis (leaves, shoots, and fruits) were collected and dried in an oven at 458 °C for 7 days. The dried parts were ground by mortar and pestle. The powder obtained (100 g) was added to 250 mL of 80% ethanol in an Erlenmeyer flask with agitation for 24 h at room temperature. The extract was filtered through filter paper (Whatman 2) in a Buchner funnel in vacuum. The filtrate was concentrated to a consistent liquid in a water bath at 40–45 °C. Concentrations of 2, 4 and 6 g L⁻¹ of the extract were prepared in water.

2.4. Plant material and surface sterilization procedure

Hypocotyl explants (approximately 5 cm length) from 3 weeks old plants of Citrullus lanatus (Thunb.) after inoculation by WMV were collected and in vitro culture procedure was used to evaluate the effect of T. orientalis on healthy and infected callus at the Tissue Culture Unit, Division of Genomic and Biotechnology, Biological Science Department, Faculty of Science-North Jeddah, King Abdulaziz University. Hypocotyls were washed by (DSDAW) three times, soaked in 70% ethanol for 1 min, and then washed thoroughly with DSDAW. Hypocotyls were transferred into 20% (v/v) sodium hypochlorite solution plus 2 drops of tween-20 as a surfactant material and kept on a shaker for 5 min. sterilized hypocotyls were rinsed three times in DSDAW under aseptic condition in a laminar air- flow hood. Under in vitro culture condition hypocotyls were divided by a simple surgical treatment under aseptic conditions.

2.5. Media and culture conditions

Hypocotyl explants were placed in three replicate plates containing 25 mL agar solidified basal MS medium (Murashige and Skoog, 1962) supplemented with 100 mg L⁻¹ Myo-inositol, 1.00 mg L⁻¹ thiamine–HCl, 2.00 mg L⁻¹ 2,4-D, 30 g L⁻¹ sucrose and different concentrations of Thuja extract at 2, 4 and 6 g L⁻¹. The pH was adjusted to 5.7 by either 1 M NaOH or HCl, prior to autoclaving at 121 °C and 15 psi for 20 min. The cultures were incubated in the dark at 25 ± 1 °C for 5 weeks to encourage callus initiation and induction (Fig. 1). After 4 weeks the friable callus fresh weight was measured (g).

Figure 1.

Samples of Citrullus lanatus (Thunb.) plants showing green mosaic; blistering, vein banding and malformation were collected from different fields of Jeddah. In leaves used for virus isolation, samples (a) show healthy watermelon leaves, (b–e) show infected watermelon leaves.

2.6. Protein analyses

2.6.1. Total soluble protein extraction

In eight treatments healthy and infected calluses under different concentrations of Thuja extracts for antiviral application were collected and ground to flour in a mortar by using liquid nitrogen. Total soluble proteins were extracted in SDS reducing buffer, (stored at room temperature) composed of Deionized water (38 mL), 0.5 M Tris–HCl pH 6.8 (10 mL), Glycerol (8 mL), 10% (w/v) SDS (16 mL), 2-mercapto-ethanol (4 mL) and 1% (w/v) Bromophenol blue (4 mL) until a total volume of 80 mL. The sample was diluted to a ratio of at least 1:4 with sample buffer and the extract was centrifuged at 10,000 rpm for 20 min. The total soluble protein in the supernatant was estimated according to the method of Bradford (1976) by using bovine serum albumin as standard protein. Protein content was adjusted to 2 mg mL⁻¹ per sample (Tucci et al., 1996). Then 10 μL of total soluble protein was taken for electrophoresis or stored at −20 °C according to (Juo and Stotzky, 1970).

2.6.2. Determination of protein fractions

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was carried out in 10% acrylamide slab gels following the system of Laemmli (1970). Separating gels were composed of 0.75 M Tris–HCl pH 8.8, 10% SDS, 0.025% of N,N,N,N-tetramethylenediamine (TEMED) and 30% ammonium persulfate. Stacking gels contained 0.57 M Tris–HCl pH 6.8, 10% SDS, 0.025% TEMED and 30% ammonium persulfate. Electrode buffer contained 0.025 M Tris, 0.192 M glycine, 0.1% SDS and pH 8.3. Electrophoresis was carried out with a current of 25 mA and 130 V per gel until the bromophenol blue marker reached the bottom of the gel after 3 h. After electrophoresis, the Coomassie Brilliant R250 staining method was used for protein bands and polypeptides.

3. Results and discussion

3.1. Watermelon mosaic virus sources, isolation and propagation

Data illustrated in Fig. 1 showed that in the naturally infected watermelon plants with WMV appeared viral symptoms of systemic mosaic, blisters, chlorotic blotching, leaf deformation, vein banding, blisters and leaf distortion. WMV was identified by Enzyme-linked immunosorbent assay (ELISA), using WMV polyclonal antibodies. Extracts from naturally virus infected watermelon leaves were found to react with WMV antibodies but not with antibodies of the cucumber mosaic virus (CMV), squash mosaic virus (SqMV) and Zucchini yellow mosaic virus (ZYMV). Samples that give a positive reaction with WMV were collected separately and used for virus propagation on watermelon seedling of C. lanatus (Thunb.). Symptoms caused by WMV on watermelon leaves appeared in a wide range of foliar discoloration and malformation symptoms such as, mosaic, blisters, chlorotic blotching, leaf deformation, vein banding, blisters and leaf distortion. This virus was isolated in previous studies from cucurbits, by other investigators in different countries (Gulsiri et al., 2003; Farag et al., 2005; Shoeibi et al., 2010; Zohreh, 2011; Aisan et al., 2012; Lima et al., 2012; Trkulja et al., 2014).

3.2. Reverse transcription-polymerase chain reaction RT-PCR

RT-PCR was used for the detection of WMV coat protein (cp) gene in infected watermelon. PCR fragment of correct size of 825 bp was amplified with the primer WMV/F and WMV/R for WMV-cp. gene. 1.7% Agarose gel electrophoresis analysis of the amplified PCR products is demonstrated in Fig. 2. However, no product was amplified from healthy watermelon plants using the same procedure. Our results are in harmony with those reported for WMV (Zohreh, 2011) and no specific product occurred for healthy material and no bands were found when the PCR assay was attempted without the initial RT step (Farag et al., 2005; Aisan et al., 2012; Trkulja et al., 2014). Shoeibi et al. (2010) showed that all WMV sequences can be placed into 6 clades. Iranian isolates fall into two distinct clades. Golestan and Mashhad isolates were grouped with isolates from Europe, Mediterranean and Australia, while the Shiraz isolate was grouped with two isolates from Japan. Genetic distance between and within groups confirmed the phylogenetic results.

Figure 2.

Electrophoresis of agarose gel showing PCR amplifications from healthy and naturally infected watermelon leaves. Lane M DNA ladder marker, Lanes 1 represent healthy watermelon leaves. Lanes 2, 3, 4 and 5 represents PCR-positive infected watermelon leaves.

3.3. Estimation of virus concentration of WMV infectivity under Thuja extracts treatments

Extracts from T. orientalis at 6 g L⁻¹ was found to inhibit multiplication of WMV by a decrease of virus concentration by ELISA reactions absorbance to 0.147 compared with healthy and infected hypocotyl explants. Lower effect on the virus was observed by 2 and 4 g L⁻¹ (0.962 and 0.735), respectively. Conversely, Thuja extract application reduced the appearance of harmful signs caused by virus development, especially when infected hypocotyls explants were treated with 6 g L⁻¹ Thuja extract. These results are in agreement with Al-Ani et al. (2010) who found that the extracts from T. orientalis and Artemisia campestris at 6 g L⁻¹ were found to inhibit the multiplication of PLRV by 81.72 and 63.6% respectively. Lower effects on the virus (57.8% and 45.7% of inhibition) were observed by the two extracts in plants generated from infected tubers. Also, Al-Ani et al. (2011) evaluated the effect of three products, Vit-org nutrient, 2-nitromethyl phenol, and the Thuja extract on the multiplication of EBMV in eggplants and they showed that the application of these products on EBMV-inoculated eggplants at 2.5 and 1 m L⁻¹, and 6 g L⁻¹ caused a reduction in ELISA reactions absorbance to 0.073, 0.091, and 0.092, respectively, which reflect a reduction in virus concentration. On the other hand, Al-Ani et al. (2011) reported that the extracts from Thuja, Tamarix and Henna plants exhibited inhibitory effects on TYLCV multiplication in tomato treated plants with protection periods of 10–12 days.

3.4. Effect of different concentrations from Thuja extract on callus morphology and fresh weight of healthy and infected hypocotyls explants

Data illustrated in Figs. 3 and 4 showed that the effectiveness of three different concentrations of Thuja extracts on callus fresh weight obtained from healthy and infected hypocotyls explants from C. lanatus (Thunb.) in the presence of WMV generally decreased the callus fresh weight of infected hypocotyl explants to 0.021 g, compared with callus fresh weight in healthy control to 0.467 g, at the same time, all fresh weights of infected hypocotyl explants were increased when treated by three different concentrations from Thuja extracts treatments compared with untreated (control infected). 6 g L⁻¹ Thuja extracts concentration treatment recorded higher increases in callus fresh weight of infected hypocotyl explants 0.41gm compared with another concentration of Thuja extract at 2 and 4 g L⁻¹ (0.02 and 0.321 g, respectively). These results were in agreement with Al-Ani et al. (2010) and Al-Ani et al. (2011).

Figure 3.

Effect of different concentrations from Thuja extracts on callus morphology of healthy and infected hypocotyl explants. H: Healthy callus, I: Infected callus, T1: 2 g L⁻¹, T2: 4 g L⁻¹, T3: 6 g L⁻¹.

Figure 4.

Effect of different concentrations from the Thuja extract on callus fresh weight of healthy and infected hypocotyl explants. H: Healthy callus, I: Infected callus, T1: 2 g L⁻¹, T2: 4 g L⁻¹, T3: 6 g L⁻¹.

Among different concentrations of Thuja extract products used against the virus, it was found that the more effective one was the Thuja extract which restricts virus multiplication. The least effect on virus multiplication was the Thuja extract as determined by ELISA-absorption at 405 nm which demonstrated protective and curative effects of these Thuja extract. The restriction of virus multiplication was found accompanying the retardation of symptoms expression on the treated plants. Similar results were obtained in previous studies concerning the use of plant extracts to manage virus disease in both animals and plants (Al Ani and Hassan, 2002, Wannang et al., 2009, 2010; Yarmolinsky et al., 2009). The antiviral activity of the Thuja extract product used in this study is connected to its components which may act directly by interaction with virus particles in the early stage of infection and block the liberation of its nucleic acid that lead finally to stop of virus multiplication.

3.5. Effect of different concentrations from Thuja extract on soluble proteins of healthy and infected hypocotyls explants

On the other hand, Figs. 5 and 6 and Table 1 showed the changes of total soluble proteins and numbers of protein bands in healthy and infected hypocotyl explants from C. lanatus (Thunb.) cultivar (Charleston Gray No. B3 USA) in the presence WMV under in vitro culture condition treated by different concentrations of the Thuja extract. WMV infection increased soluble protein and number of protein bands of infected hypocotyl explants to 31.492 mg g⁻¹ and 14 bands, compared with the healthy control 16.49167 mg g⁻¹ and 7 bands, respectively. On the other hand, soluble protein numbers of protein bands of infected hypocotyl explants were decreased when treated by three different concentrations of Thuja extracts treatments compared with the control treatment. The increased number of bands in infected hypocotyl explants compared with healthy control is associated with denature of the enzymes involved in amino acids and protein synthesis under biotic and a biotic stress. 6 g L⁻¹ Thuja extract concentration treatment recorded higher decreased of soluble proteins of infected hypocotyl explants 16.66 mg g⁻¹ compared with the infected and another treated with concentration from Thuja extract at 2 and 4 g L⁻¹ was recorded as 30.517 and 30.72467 mg g⁻¹, respectively, compared with the control infected .These results agreement with (Al Ani and Hassan, 2002; Al-Ani et al., 2002, 2011; Al-Azawi et al., 2008; Yarmolinsky et al., 2009).

Figure 5.

Effect of different concentrations from the Thuja extracts on soluble proteins (mg g⁻¹ fresh weight) of healthy and infected hypocotyl explants. H: Healthy callus, I: Infected callus, T1: 2 g L⁻¹, T2: 4 g L⁻¹, T3: 6 g L⁻¹.

Figure 6.

SDS–PAGE profile showing the changes in protein patterns of callus from healthy and infected hypocotyls of watermelon with WMV under explants of different concentrations from Thuja extracts. (A) The protein profiling image of the SDS–PAGE electrophoresis M. protein ladder marker, L1. Infected, L2. Healthy, L3. Infected & T1, L4. Infected & T2, L5. Infected & T3, L6. Healthy & T1, L7. Healthy & T2 and L8. Healthy & T3. (B) The protein profiling peaks of the SDS–PAGE electrophoresis. M. protein ladder marker, H: Healthy callus, I: Infected callus, T1: 2 g L⁻¹, T2: 4 g L⁻¹, T3: 6 g L⁻¹.

Table 1a.

Effect of different concentrations treatments from the Thuja extract on protein bands in infected watermelon cultivar after 21 days from inoculation by WMV.

Bands No. From 1 to 25	MW KDa	RF	Present and absent protein bands
			I	I&T1	I&T2	I&T3	H	H&T1	H&T2	H&T3
1	277	0.143	+	−	−	−	−	−	−	−
2	270	0.148	−	−	+	−	−	−	−	−
3	217	0.192	+	−	+	−	−	−	−	−
4	215	0.194	−	−	−	+	−	−	−	+
5	205	0.203	−	−	−	−	−	−	+	−
6	199	0.209	−	−	−	−	−	+	−	−
7	180	0.320	+	−	−	−	−	−	−	−
8	177	0.234	−	−	−	−	−	−	+	−
9	171	0.241	−	−	−	−	−	−	−	+
10	161	0.253	−	−	−	+	−	−	−	−
11	154	0.262	+	−	−	−	−	−	−	−
12	129	0.300	−	−	−	−	−	+	−	−
13	120	0.321	−	−	−	+	−	−	−	−
14	116	0.331	−	−	−	−	−	−	−	−
15	112	0.342	−	−	−	−	−	−	−	+
16	108	0.344	−	−	−	−	+	−	−	−
17	106	0.346	+	−	+	−	−	−	−	−
18	98	0.361	−	−	−	+	−	−	−	−
19	97	0.390	−	−	−	−	−	−	−	−
20	94	0.421	−	−	−	−	−	−	−	−
21	88	0.455	+	−	−	−	−	+	+
22	83	0.467	−	−	−	−	−	−	−	+
23	77	0.477	−	+	+	−	−	−	−	−
24	72	0.478	+	−	−	−	−	−		−
25	69	0.481	−	−	−	−	−	−	+	−

Table 1b.

Effect of different concentrations treatments from the Thuja extract on protein bands in infected watermelon cultivar after 21 days from inoculation by WMV.

Bands No. From 26 to 50	MW KDa	RF	Present and absent protein bands
			I	I&T1	I&T2	I&T3	H	H&T1	H&T2	H&T3
26	68	0.483	−	−	−	−	−	+	−	−
27	66	0.484	−	+		+	−	−	−	−
28	65	0.486	−	−	+	−	−	−	−	+
29	59	0.487	+	−	−	−	−	−	−	−
30	57	0.498	−	−	−	−	−	+	+	−
31	51	0.499	−	+	−	−	+	−	−	−
32	48	0.549	+	−	−	−	−	−	−	−
33	47	0.559	−	−	+	−	−	−	−	−
34	46	0.565	−	−	−	+	−	−	+	+
35	45	0.568	−	−	−	−	−	−		−
36	42	0.587	+	+	−	−	−	+	+	−
37	40	0.589	−	−	−	−	+	−	−	−
38	38	0.633	−	−	−	−		−	−	+
39	36	0.673	−	+	−	−	−	−	−	−
40	32	0.707	−	−	−	+	−	−	−	−
41	31	0.726	+	−	−	−	−	−	−	−
42	30	0.771	−	−	−	+	−	−	−	−
43	27	0.783	+	+	+	−	−	−	−	+
44	26	0.842	−	−	−	−	+		+	+
45	25	0.874	+	−	+	−	+	−	−	−
46	24	0.891	−	+	−	+	−	+	+	−
47	23	0.963	+	+	+	−	+	−	−	+
48	22	0.974	−	+	−	−	+	−	−	−
49	21.5	0.981	−	−	−	−	−	−	−	−
50	14	0.995	−	−	−	−	−	−	−	−

4. Conclusion

WMV is a major limiting factor for the production of watermelon worldwide. For the effective control of this virus by using some plants extracts such as T. orientalis extracts, three different concentrations at 2, 4 and 6 g L⁻¹ were applied under in vitro culture conditions. 6 g L⁻¹ of Thuja extract concentration followed by 4 g L⁻¹ was more effective to reduce the infection of WMV compared with g L⁻¹ or control treatment either in healthy or infected hypocotyl explants. It is evident from this study that the reverse transcription-PCR assay is important since it is relatively easy to obtain valuable data and it can be useful to detect the WMV coat protein (cp) gene in infected watermelon. Also, in infected hypocotyl explants treated with 6 g L⁻¹ the increase in the number of soluble protein bands which are synthesized specifically or at a higher rate of infection compared with other treatments may play an adaptive role in plant during infection, protecting the key cytoplasmic enzymes and protein synthesizing apparatus against the adverse effect of WMV.