Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace

Abstract Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV‐infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV‐infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus‐free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus‐free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus‐free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.

diseases such as cassava mosaic disease (CMD) (Bull, Ndunguru, Gruissem, Beeching, & Vanderschuren, 2011). CMD is caused by at least nine species of cassava mosaic begomoviruses (CMBs) that can be broadly categorized as African cassava mosaic viruses (ACMVs) and East African cassava mosaic viruses (EACMVs). These viruses are transmitted by whiteflies (Bemisia tabaci Gennadius) and spread through infected planting materials and contaminated implements, and their existence can cause yield losses of up to 100% on susceptible genotypes (Varma & Malathi, 2003).
Virus elimination is a technique used for the production of virusfree planting materials. There are several virus elimination methods, including thermotherapy, chemotherapy, electrotherapy, somatic embryogenesis and meristem tip culture (Panattoni, Luvisi, & Triolo, 2013). These methods can be used alone or in combination with other methods to obtain virus-free plantlets (Paprstein et al., 2008).
There have been reports of successful elimination of viruses from infected plants through chemotherapy and thermotherapy, for example Hu et al. (2015). In another study, chemotherapy produced 100% virus-free grapevine plants with simple and mixed infection (Guta, Buciumeanu, Gheorghe, & Al, 2014). Furthermore, the elimination of Cassava brown streak virus from cassava was achieved by combining chemotherapy and thermotherapy in Kenya (Mwagangi, Ateka, Nyende, & Kagungu, 2014). However, currently efficacy of chemotherapy and thermotherapy in eliminating viruses causing CMD from Tanzanian landraces is unknown. The aim of this study was to evaluate the effectiveness of chemotherapy and thermotherapy in eliminating EACMV in widely grown, farmer-preferred cassava landraces in Tanzania.

| Source of plants and screening for the presence of cassava mosaic begomoviruses
Twenty stem cuttings of the cassava cultivar Kibandameno showing CMD symptoms were collected from Tanga region in Tanzania, established and maintained in a greenhouse at Mikocheni Agricultural Research Institute. Ten days after sprouting, leaves were collected and used for DNA extraction using the cetyl trimethyl ammonium bromide (CTAB) method (Lodhi, Ye, Weeden, & Reisch, 1994;Xu, Aileni, Abbagani, & Zhang, 2010).
The extracted DNA was subjected to polymerase chain reaction using the primer pair EAB555, forward (5′-TACATCGG CCTTTGAGTCGCATGG-3′) and reverse (5′-CTTATTAACGCCT ATATAAACACC-3′) that amplifies approximately 555 bp nucleotides. The PCR reaction mix contained 17.3 μl of sterile distilled water, 1 μl dNTPs (2.5 mM), 2.5 μl ×10 PCR buffer + 20 mM MgCl 2 , 0.2 μl Taq polymerase, 1.0 μl of primer mix and 2 μl of DNA template. PCR conditions were as follows; predenaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min and elongation at 72°C for 2 min and final elongation at 72°C for 10 min. Samples were then stored at 4°C until used for further analysis. PCR amplification was checked by loading 5 μl of PCR products in 1.5% agarose gel stained with ethidium bromide submerged in 1X TE buffer for 50 min at 180 V.
The PCR products were visualized on a gel documentation system (BioDoc-It ® 210, USA) and photographed.

| Multiplication of EACMV positive cassava plants
Murashige and Skoog medium (MS) supplemented with 20 g/L sucrose and 3 g/L plant agar was sterilized at 121°C for 15 min before being used for initiation and multiplication of EACMV-infected cassava plants.
Nodal cuttings of EACMV-infected cassava plants were taken using a clean surgical blade and samples were washed vigorously with tap water containing detergent (Tarmol ® ) and three drops of Tween-20 to achieve surface sterilization. The explant was then taken to a safety hood and washed with 70% ethanol for 5 min.
They were subsequently rinsed in sterile distilled water three times.
The explants were then soaked in sodium hypochlorite containing Tween-20 for 2 min and then rinsed with sterile distilled water until foam had been removed. The ends of the nodes were cut with a sterile surgical blade, grown on MS medium and incubated in a growth room at 28°C with a photoperiod of 16 hr light and 8 hr dark.
Subculturing was performed after 5 weeks.

| Chemotherapy
Salicylic acid and ribavirin were used as antiviral compounds. They were prepared and filter-sterilized using a 0.22 μm Millipore filters.
Nodal cuttings from infected cassava were grown in MS medium supplemented with salicylic acid at concentrations of 0, 10, 20, 30 and 40 mg/L and ribavirin at concentrations of 0, 5, 10, 15 and F I G U R E 1 Detection of EACMVinfected cassava. Lane 1, 1 kb molecular marker; lane 2 and lane 3, are positive and negative control, respectively. Lane numbers 1 to 15 correspond to the infected cassava plants that were tested 20 mg/L for three weeks at 28°C in a photoperiod of 16 hr light and 8 hrr dark. After 3 weeks the plants were transferred into medium without antiviral compounds before PCR analysis was performed.

| Thermotherapy
Infected nodal cuttings from subcultured plants were grown in MS media in a tissue culture growth room at 28°C for 2 weeks and then moved to a heat chamber with 16 hr light and 8 hr dark for 3 weeks.

| Experimental design, data collection and analysis
The chemotherapy experiment had two treatments, composed of salicylic acid and ribavirin at five concentration levels for each. Each

| RE SULTS
The PCR products from tested plants were visualized in agarose gel.
Of the leaf samples tested, 13 gave PCR products using EACMVspecific primers while 2 did not amplify ( Figure 1). The EACMV positive plants were cultured and used for thermotherapy and chemotherapy treatment.

| Effect of ribavirin on survival and growth of in vitro cassava plants
A total of 123 out of 192 cassava plants survived treatments with ribavirin at different concentrations (Table 1) Table 1.  Plants did not survive the highest concentration of salicylic acid (Table 2).  Figure 4c). Survival % among the four temperature regimes was over 47% (Table 3). Thermotherapy treated plants at higher temperature resulted in lower survival % of 47.9% at 40°C compared to 93.8% at 30°C.

| Effect of thermotherapy on growth and survival of in vitro cassava plants
Survival decreased with increase in temperature ( Table 3).

| Efficiency of chemotherapy and thermotherapy in elimination of EACMV from infected cassava plants
The results obtained showed that virus elimination efficiency relied on chemical concentration and temperature regime. Higher concentrations and higher temperatures resulted in greater levels of virus elimination. In the 20 mg/L ribavirin treatment, 85% of plantlets were virus-free, while at 5 mg/L, this figure was 52% (Table 1).
Thermotherapy resulted in more than 50% virus-free plants with 35°C showing the highest virus elimination of 79.5%. At 40°C, the number of surviving plantlets was observed to be low but the efficiency of virus elimination was high (69.5%) compared to 30°C which showed 48.9% (Table 3). of regenerated clean plants as well as low survival % were found for both ribavirin and salicylic acid-treated plants at high concentrations (Table 1 and 2). At this point viral RNA mutation exceeds a critical threshold and leads to infectivity and/or extinction of the virus population (Panattoni et al., 2013). On the other hand, a high plant regeneration rate occurred at low concentrations since these levels were less toxic and furthermore, over 50% were virus-free.

| D ISCUSS I ON
These results showed that even at low concentrations virus replication was hindered as in the cases of ribavirin at 5 mg/L and salicylic acid 10 mg/L (Table 1 and 2). Similar results were obtained by Seker, Süzerer, Elibuyuk, and Çiftçi (2015) in eliminating Plum pox potyvirus (PPV) from infected apricot shoots using chemotherapy.
Even though salicylic acid-treated plants had a lower survival % at higher concentration of the chemical, treatment resulted in a greater percentage of virus-free plants. This result is consistent with that of Mwagangi et al. (2014) where they note a similar effect after chemical treatment.
Regarding temperature, different regimes affected the efficiency of virus elimination from infected cassava plants.
Thermotherapy has been widely used to eliminate viruses from trees, herbaceous plants and other vegetative plants (Panattoni et al., 2013;Tan, Wang, Hong, & Wang, 2010;Valero, Ibanez, & Morte, 2003). In recent studies addressing the use of RNA to destroy viruses no relationship was reported between RNA silencing and temperature. Plant-virus interaction is affected by temperature and higher temperatures are associated with minimal virus levels in infected plants (Qu et al., 2005). Wang

| CON CLUS ION
This study demonstrated that virus-free plantlets can be produced in

This work was supported by the Bill and Melinda Gates foundation and
Mikocheni Agricultural Research Institute, Dar es Salaam Tanzania.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as potential conflict of interest.