Inhibition of Rehmannia mosaic virus Infection by Ganoderma sp. Extract

In fruiting bodies and mycelia of several fungi belonging to a Basidiomycetes group, among others, Ganoderma contains active polysaccharides and has potential as an antiviral substances. This study aimed to determine the effect of mycelium and fruiting body extract of Ganoderma sp. against Rehmannia mosaic virus (ReMV) infections on Chenopodium amaranticolor with variations of dilution and application times. The results showed that mycelium and fruiting body extract of Ganoderma sp. could inhibit ReMV infections. Antiviral extract which were applied 4 hours before, and at the same time with virus inoculation resulted in a higher viral inhibition rates than when applied 4 hours after virus inoculation. Fruiting body extract at 10-1 and 10-2 dilutions delayed symptom appearance caused by ReMV infection when applied 4 hours before virus inoculation. Mycelium extract at dilution of 10-2 and 10-3 applied 4 hours before virus inoculation resulted in the highest inhibition rate (100%) which was indicated as the number of local lesions on Chenopodium amaranticolor leaves. Meanwhile the fruiting body extract dilution of 10-1 resulted in highest inhibition (99.55%) when applied 4 hours before virus inoculation.


INTRODUCTION
Rehmannia mosaic virus (ReMV) was first reported in Henan, China as a virus of Rehmannia glutinosa Libosch. Symptoms caused by this virus is a systemic mosaic similar to symptoms caused by Tobacco mosaic virus. ReMV can be transmitted mechanically on Nicotiana glutinosa and result in necrotic lesions. In addition, this virus also causes the same symptoms on Nicotiana tabacum var. Xanthi, N. rustica, Datura stramonium and Chenopodium amaranticolor (Zhang et al., 2008). This virus was first reported in Japan on chili pepper variety Fushimi-amanaga. Symptoms from this infection are mosaic symptoms on leaves which continuously develop and cause leaves to fall, necrotic lesions on stems, and malformed fruit (Kubota et al., 2012).
According to Endarsih et al. (2017), Rehmannia mosaic virus are known as the cause of systemic mosaics on tobacco. Symptoms caused by this infection are leaf malformations and systemic mosaics. This virus causes variety of symptoms on several plants. Chlorotic systemic lesions were shown on cucumber plants. Symptoms on C. amaranticolor are chlorotic and local necrotic lesions, while on chili pepper, tobacco, and eggplants, symptoms found was systemic mosaics (Nurviani, 2018). Therefore, this virus has a wide host range and can easily be transmitted; thus, there is a need for an antiviral compound to manage this virus. Basidiomycota is a phylum recognized to contain many natural antibiotic compounds. Some species have been reported to possess antifungal and antiviral compounds, inhibit phytopathogenic nematodes and myeloma cell growth, as well recognized as antioxidants (Somowiyarjo, 1999;Sokovic et al., 2013;Sivanandhan et al., 2017).
Indonesia has high biodiversity of fungal species, such as the genera Ganoderma, which are still unknown of their practical use (Tampubolon et al., 2013). Some species are known as plant pathogens of several important plantation crops (Semangun, 2000;Purnamasari et al., 2012). However, research by Kovalenko et al. (2008) reported that metabolites produced by G. lucidum and G. applanatum were able to inhibit Tobacco mosaic virus infection by 65-70% at the concentration of 1000 µg/mL This goal of this study was to determine the antiviral potential of mycelium and fruiting body of Ganoderma sp. against Rehmannia mosaic virus infection.
Observation in this study included (1) symptoms and virus incubation period done every day for 10 days after C. amaranticolor leaf inoculation; (2) number of local lesions on each treatment, which were done every day for 10 days after C. amaranticolor leaf inoculation; (3) inhibition percentage of ReMV infection on test plants that were calculated using the following formula (Lu et al., 2013): Average local lesions and inhibition rates were analyzed using ANOVA. If significant differences were found, a Duncan post hoc test (DMRT) was conducted. All tests were conducted at α=0.05.

Disease Symptoms and Incubation Period
Local lesions appeared on C. amaranticolor leaf inoculated with ReMV ( Figure 1). The symptoms started with yellow lesions (Figure 1.b), which enlarged causing several small lesions forming a larger one and turned brownish 7 days after inoculation (Figure 1.c). Leaf areas around the lesions turned yellow followed by death of the leaf causing leaf to fall (Figure 1.d).
Incubation period of the virus is shown in Figure 2. ReMV had an incubation period of 4 days, which was implied by observations on leaves inoculated with virus without antiviral treatment (positive

Test Plant Preparation
Test plants used in this study were approximately 2months-old C. amaranticolor that were grown in dark conditions 24 hours prior to inoculation.

Ganoderma sp. Isolation and Propagation
Ganoderma sp. used in this study was obtained from a river tamarind (Leucaena leucocephala) tree located on Kalimantan Street, Sinduadi, Sleman, Special Region of Yogyakarta. Fungus was isolated and the growing mycelia were then cultured on a new PDA medium. This later was stored as a pure culture for subsequent tests.

Ganoderma sp. Extract Preparation
Ganoderma sp. extract was prepared from mycelia and fruiting body. Mycelia that were dried in an oven, weighted, macerated using a porcelain mortar, and sterile water was added to obtained an extract concentration of 1 g/mL. Ganoderma sp. extract from fruiting body was obtained by drying macerated fruiting body, weighting and adding sterile water to obtain extract concentration of 1 g/mL.

Virus Inoculation and Propagation
Virus isolate used in this study was obtained from tobacco plants with mosaic symptoms located at Kedu, Temanggung, Central Java. Virus were then propagated on tobacco plants and molecularly identified using TobRT-up1 and TobRT-do2 primer pairs. Sequence results confirmed that the virus isolate had 97% homology with Rehmannia mosaic virus (ReMV) of Capsicum annuum Japan isolate (Endarsih et al., 2017).
Virus inoculation was conducted using a sap substance made by adding 10 mL of phosphate buffer 0,01M pH 7 to 1 g of macerated infected tobacco leaves. Virus sap subtance was diluted to 10 -4 and was inoculated by damaging plant leaves with rubbing 600 mesh of carborundum and followed by rubbing virus sap subtance. Leaves were cleaned using sterile water after inoculated with virus sap subtance.

Ganoderma sp. Extract as an Antiviral Agent
Both Ganoderma sp. extract types were diluted to 10, 100, and 1000 times using sterile water. Each dilution series were applied 4 hours before, 4 hours after, and same time as virus inoculation on test plants with a ratio of 1:1 between virus sap substance and Ganoderma sp. extract. This study was designed with a complete randomized design with 5 replications for each treatment combination. Application was conducted by rubbing Ganoderma extract on leaf surface of C. amaranticolor with the following treatments:  Antiviral application together with virus inoculation at 10x dilution showed similar incubation period (Figure 2.b). Antiviral treatment using mycelia extract 4 hours before inoculation with dilution of 100× and 1000× did not show symptoms until observation were finished (10 days after inoculation) (Figure 2.a).

Ganoderma sp. Extract as an Antiviral Substance against ReMV
The effects of mycelial extract against ReMV infection is shown in Table 1. Positive control showed the greatest number of local lesions compared to leaves treated with antiviral substance. Antiviral substance applied 4 hours before inoculation resulted in the least number of lesions compared to other application time treatments, whereas antiviral applied 4 hours after inoculation showed the highest number of lesions. The effect Ganoderma sp. fruiting body extract against ReMV (Table 1) showed that the highest number of lesions was the positive control (44 lesions). Antiviral substance applied 4 hours before and same time as virus inoculations showed relatively fewer lesions compared to antiviral substance applied 4 hours after inoculation. Dilution rates corresponded positively with the number of lesions.

ReMV Inhibition Rate of Ganoderma sp. Extracts
Mycelium extract was able to inhibit up to 100% with an average number of local lesions of 0, while fruiting body extract showed the highest inhibition rate of 99.55% with an average 0.2 lesions (Figure 3). The both highest inhibition rates were obtained from application times of 4 hours before inoculation. Antiviral substance application before virus inoculation tend to show higher inhibition rates compared to other application times. This was shown by the fewer numbers of lesions. According to Awasthi et al. (2016), there are 3 mechanism that may occur when virus infections are inhibit, such as (1) direct effects on virus by non-activating or alternating virus characteristics; (2) effects on infectious process of viruses, especially ones that are highly related to ribosomal inactivating protein (RIPs), by hindering virus replications and protein synthesis of host ribosomal cells; and (3) affecting host susceptibility by changing cell metabolism and producing antiviral substance. The later mechanism in an indirect mechanism, which frequently occurs as induced local and systemic resistance mechanisms.
For analytical purposes, data were transformed using √(x+0.5). Antiviral substance application together with virus inoculation showed decent results. Infection inhibition rates were positively correlated to dilution rates of antiviral substance. According to Rini (2004), higher dilution rates caused lower effectivity in suppressing local lesions resulted from this infection.
Application time of antiviral substance after virus inoculation resulted in lower inhibition rates. According to Narwal et al. (2001), antiviral application after virus inoculation were more effective when applications were done immediately after virus inoculation. Inhibition rates will decrease over time as time between virus inoculation and antiviral substance application increases. The same research showed that antiviral substance application 4 hours after inoculations were only able to inhibit up to 46%. Furthermore, antiviral substance application after inoculation was not able to eliminate virus. This may imply that this antiviral may not have chemotherapeutic mechanism as shown by the use of ribavirin in the research by Roostika et al. (2016) which proven to be able to eliminate Sugarcane streak mosaic virus (SCSMV) on sugarcane variety PS862-Crb.
In G. lucidum, polysaccharides were the main components and more than 200 polysaccharides have been isolated from this species where most of them were β-glucan. Several researches have shown that β-glucan has antiviral activities by hindering virus penetration and replications (Minari et al., 2011;Rouhier et al., 1995). Therefore, β-glucan might be a component with antiviral substance in Ganoderma that may inhibit ReMV infection.

CONCLUSION
Mycelia and fruiting body extract of Ganoderma sp. possessed antiviral properties against ReMV infection. Mycelial extract at 10 -2 and 10 -3 applied 4 hours before virus inoculation resulted in the highest inhibition of 100%, while fruiting body extract at dilution rate of 10 -1 resulted in the highest inhibition of 99.55% when applied 4 hours after inoculated with virus sap substance.