MATERIALS AND METHODS

Source of virus: Samples from naturally infected potato plants exhibiting viral infection were collected from Al-Behera Governorate and directly transferred to the laboratory for detection. The observed symptoms included mild mosaic and crinkle on leaves.

Isolation and identification
Selective host plants and symptomatology: About 2 g of naturally infected potato leaf tissues were grounded in 0.01 M phosphate buffer, containing 0.2% Diethyldithiocarbamate, at pH 7.2 then, mechanically transmitted to each three seedlings of the following host plant, Gomphrena globosa L., Chenopodium amaranticolor L. and Chenopodium quinoa wild grown in clay pots containing sterilized soil and kept in an insect-proof greenhouse. Four weeks later, seedlings were examined for symptoms expression by visual inspection and DAS-ELISA.

The virus isolate was biologically purified from the single local lesion as reported by Kuhn (1964) produced on G. globosa. After successive signal local lesion transfers in the local lesion host, the resulting virus isolate was propagated in N. tabacum cv. White Burley plants.

The sap from systemically PVX-infected N. tabacum cv. White Burley were inoculated onto healthy potato leaves slightly dusted with carborundum 600 mesh and served as a source for in vitro and in vivo experiments.

Serological detection: Enzyme linked immune-sorbent assay (DAS-ELISA) was conducted to test the isolated virus against PVX according to Clark and Adams (1977). Observation was measured by ELISA-Reader at 405 nm. Positive result signed if the tested samples had average point more than 2x point of negative sample. All tests were carried out in the serological laboratory of Virus and Phytoplasma Research Department, Plant Pathology Research Institute, ARC. Plants reacted positively against PVX antiserum were served as source of virus inoculums.

Effect of lactoferrin on PVX in vitro: Explants of 2-3 cm in length were collected from one month old potato plants after inoculation that being maintained in the greenhouse and then were surface sterilized with 20% commercial Clorox solution and one drop of tween 20 and left for 20 min then rinsed 3 times with sterile distilled water for 5 min each.

The surface sterilized explants were cut into single nodal segments and cultured in sterilized culture jar containing 25 mL modified solid MS medium according to Murashige and Skoog (1962), supplemented with 30 g L^-1 sucrose and 9 g L^-1 agar without any additions of growth regulators and then kept in a growth chamber. The established in vitro plantlets were then sub-cultured every three weeks using single node cuttings to build up the stock plants necessary for the experiment. Four levels of lactoferrin powder 100, 250, 500 and 1000 mg were applied and solid medium was replaced with liquid one. Each level was dissolved in 100 mL of autoclaved liquid culture medium and filter sterilized before adding to 900 mL of the culture medium inside a laminar flow, then left for 48 h. Ten infected plantlets with PVX were sub-cultured on filter paper bridge in liquid medium supplemented with each level of lactoferrin. The percentage of virus-free plantlets was indexed using DAS-ELISA test after 21 days.

A complete randomize design was used for analysis all data with three replications. The treatment means were compared by least significant difference (L.S.D) test as given by Snedecor and Cochran (1994).

All experiments were conducted under aseptic condition in laminar flow and cultures at all growth stages were incubated under artificial conditions 22/18°C day/night temperatures and 16 h photoperiod for three weeks.

Effect of lactoferrin on PVX in vivo
Pre-inoculation treatment: Each concentration of lactoferrin was sprayed on the tested plants which were mechanically inoculated separately with PVX infected sap at different intervals: 1, 3, 5 and 7 days, respectively. Distilled water was used as a control. Twenty plants were used for each treatment. The inhibition percentages of virus were assessed firstly on the basis of symptom expression and then examined using DAS-ELISA.

Post-inoculation treatment: In this experiment, virus infected sap was applied first followed by lactoferrin treatment after 7 days. Distilled water was used as a control. Each treatment was conducted twice and twenty plants were used for each treatment. Samples of leaves were collected from each treatment separately after 4 weeks from inoculation, then examined using DAS- ELISA to determine the antiviral activity of lactoferrin against PVX.

RESULTS AND DISCUSSION

Isolation and identification: As shown in Fig. 1a samples collected from naturally infected potato plants showed mild mosaic or crinkle on potato leaves which is in accordance with symptoms previously described for PVX infection on potato plants (Muhammad et al., 2012).

Sap of naturally infected potato leaf tissues were mechanically inoculated into tested plants. After biological purification, the virus isolate was re-inoculated onto the selective host plants. Three plant species belonging to two families (Amaranthaceae and Chenopdiaceae) were mechanically inoculated with PVX, showed only local lesions on Gompherena globosa L. after one month and serologically reacted against PVX antiserum while, no symptoms appeared on Chenopdiaceae family (Chenopodium amaranticolor and Chenopodium quinoa) and no serological reaction was detected against PVX antiserum. The local lesion host of PVX was propagated in Nicotiana tabacum cv. White Burley plants and showed mosaic on leaves after one month of incubation period under greenhouse conditions. Similar results were also recorded by El-Araby et al. (2009). The previously obtained PVX was mechanically transmitted to healthy potato plants (Fig. 1b) showed mosaic and crinkle on leaves (Fig. 1c and d).

Therapeutic effect of lactoferrin against PVX: Experiments were conducted in vitro and in vivo. The goal of in vitro phase of our study is to simulate the vegetative propagation process which is used to commercially production of potato plantlets throughout the year and to identify the most effective lactoferrin concentration on the virus-free plantlets production or the growth of plant as well as to evaluate the potential antiviral effects of different concentrations of lactoferrin by spraying the inoculated potato plants.

It was clear that the treatment with concentration 1000 mg L^-1 of lactoferrin is more distinguished for antiviral activity as compared to the other concentrations which increased the percentages of PVX-free potato plantlets to 80% in vitro (Table 1) or 70 and 50% under greenhouse condition (Table 2) with significant differences between treatments (lactoferrin concentrations). Also, the in vitro treatment was of high efficiency when compared with lactoferrin based-spraying which may be due to the loss of some amount of dissolved lactoferrin in the air.

Fig. 1(a-d):	(a) Naturally infected potato plants, (b) Healthy potato plants, (c) PVX-developed mosaic symptom and (d) Crinkle on leaves after mechanical inoculation of potato plants under greenhouse condition

Table 1:	Effect of lactoferrin on production of PVX-free plantlets in vitro
Ten plants/treatment-Data is based on DAS-ELISA detection

Table 2:	Effect of lactoferrin treatments on post-inoculated potato plants by PVX under greenhouse condition
Twenty plants/treatment-Data is based on DAS-ELISA detection

Table 3:	Effect of pre-treatments of lactoferrin on PVX infectivity in vivo
Twenty plants/treatment-Data is based on DAS-ELISA detection

The antiviral activity of lactoferrin or its lobes against intracellular viral particles is still unclear. Generally, lactoferrin is a polypeptide chain and a number of studies have implicated these peptides that mainly derived from the N-lobe, to be responsible for antiviral role through its ability to interact with the viral molecules (Siciliano et al., 1999) or based on the protein cationicity and α-helical structure of lactoferrin (Lin et al., 2011; Zhang et al., 2013). Furthermore, lactoferrin has been described as an antiviral agent that affects a broad range of RNA and DNA viruses that infect humans and animals (Gonzalez-Chavez et al., 2009) and plays a central role in the immune system of the body; in spite of plant systems are far less likely to harbor microbes pathogenic to humans than mammalian cells but one of the major advantages of plants is that they possess an endomembrane system and secretory pathway that are similar to mammalian cells (Vitale and Pedrazzini, 2005).

These hypothesizes confirm the results of DAS-ELISA assays to monitor the effect of anti-PVX therapy supported the therapeutic evaluation of lactoferrin on the in vitro PVX that may be explains the antiviral activity in the current study through application of lactoferrin in culture media as evident by no variation or change in the percentages of healthy potato plants after the first therapeutical trials or reconfirmed one. Finally, it is worthy to note that lactoferrin must be added after autoclaving the culture medium through sterilized filter to avoid losing of its antiviral activity by heat (Van der Strate et al., 2001).

Preventive effect of lactoferrin against PVX: The goal of this part of study is to decrease the injury of infected potato plants through spraying different concentrations of lactoferrin under greenhouse condition. Data presented in Table 3 showed also that the most effective pre-inoculation treatment was 1000 mg L^-1 of lactoferrin as compared to the other concentrations. It is clear that the preventive treatment based on spray to be necessary every 3 or 5 days which lead to increase the inhibition percentages to 85 or 90%, respectively with significant differences between lactoferrin concentrations. A negative reaction after 7 days where all inhibition percentages reduced again in each treatment even concentration 1000 mg L^-1 of lactoferrin.

According to the literatures, techniques using milk are frequently used in nurseries to stop the spread of virus between susceptible hosts when people touch the plant, during pruning and the inhibitory effects of milk were restricted by reducing the plant’s susceptibility to the virus. These effects were on the virus and not on the plant (Gillian, 2005). Different modes of action of milk-based sprays were provided by Bettiol (1999), included an increase in the pH of the leaf surface (Ziv and Zitter, 1992), the establishment of a protective barrier (Mucharromah and Kuc, 1991; McGrath and Shishkoff, 1999) and the direct induction of systemic resistance (Reuveni et al., 1993). Many studies have shown that at least part of the antiviral properties of milk can be attributed to a direct antiviral activity of lactoferrin (Abdelbacki et al., 2010).

The interpretation of the application of lactoferrin to control viral infection is based on preventing virus entry into cells by the fact that it binds to the envelope virus protein (Yi et al., 1997) or binds to cell-surface molecules that virus use either as receptors or co-receptors (Van der Strate et al., 2001). The interaction of lactoferrin with viral envelope proteins or with receptors on cell surface is critical to blocking viral entry to target cells and infection is stopped at an early stage (Ward et al., 2005). Therefore, lactoferrin not only prevents infection but also induces some type of resistance that maintains antiviral effects after the virus has entered the cell and inhibits the proliferation or replication of viruses (Abdelbacki et al., 2010).

Effect of lactoferrin on plant growth: Increasing the concentration of lactoferrin had a positive influence on the performance proliferation of potato plantlets or could induce different multiple shooting responses without affecting the survival percentage where best results were obtained with MS media containing 1000 mg L^-1 of lactoferrin due to the possession of activities of cytokines or modulates a variety of cellular functions and increase cytokine response (Wakabayashi et al., 2004). So, it is capable to penetrate a cell and speed up the process of cell division and thus has a profound effect on shoot formation or the proliferation of cells and act as growth factor activator "growth regulator" (Yanaihara et al., 2000).

CONCLUSION

Lactoferrin seemed to be very successful and inexpensive in controlling viral infection through in vitro technique or by spraying the infected and/or healthy plant under greenhouse condition and addition of lactoferrin in the culture media to be effective for enhancing the development of cultured explants. So it can be concluded that more attention must be focus on lactoferrin with high level (1000 mg L^-1) in several crops through tissue culture technique to optimize regeneration of plantlets. Moreover, lactoferrin is a natural compound that allowing safe use based-spray in greenhouse cultivation thus avoiding the use of chemical compounds to immunize and protect plants against viral infection.

ACKNOWLEDGMENT

The authors thank Armor Proteins (France) for their kindly provided offers.