Double Infection of Onion yellow dwarf virus and Shallot latent virus in Garlic from Several Regions in Indonesia

Viruses have been a problem on garlic cultivations in various countries. There are several viruses reported infecting garlic. Genera Potyvirus and Carlavirus are the most common viruses found infecting garlic. Mixed infection on garlic is often designated as a “garlic viral complex”. These viruses can be transmitted through imported garlic seeds. Therefore, it is necessary to conduct early detection of garlic seeds to prevent the epidemic of these viruses. This study aimed to detect Onion yellow dwarf virus (OYDV) and Shallot latent virus (SLV) on garlic. Garlic samples were obtained from Enrekang, Magelang, Temanggung, Tawangmangu, and Yogyakarta. Total RNA was extracted from the samples and subsequently used for RT-PCR using two pairs of specific primers SLV-F/SLV-R and OYDV-F/OYDV-R. Primary pair SLV-F/SLV-R in amplicons sized 276 bp, while OYDV-F/OYDV-R in amplicons sized 112 bp. RT-PCR results showed that OYDV was found in all samples tested in this study. Meanwhile, double infections (OYDV and SLV) were found in eight out of ten samples tested. These results indicated that double infections on garlic were common in Indonesia.


INTRODUCTION
Garlic is an agamic crop, vegetatively propagated by bulbs over many centuries. Vegetative propagation has become the main mechanism of transmission of garlic viruses. This condition is believed to be linked to the vegetative propagation has resulted in it being infected by many virus species. Infection by many virus species is often designated as 'garlic viral complex' (Celli et al., 2016). Therefore, multiple infections are common and leading to result in yield losses and varietal degeneration (Nam et al., 2015;Pramesh & Baranwal, 2015). There are several viruses reported infecting garlic belong to genera Potyvirus and Carlavirus. Species on Potyvirus genus reported infecting garlic such as Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), and Shallot yellow stripe virus (SYSV) (Hu et al., 2015;Swari et al., 2015). Meanwhile, species belonging to the Carlavirus genus comprise Garlic common latent virus (GarCLV), Garlic latent virus (GLV), and Shallot latent virus (SLV) (Da Silva et al., 2019).
The viruses can induce both symptomatic and asymptomatic, indicating that latent infection is common. However, there is a constraint on virus detection based on visual symptoms only (Abraham et al., 2019). Therefore, detection methods with high accuracy are deployed, including serological-based and nucleotide-based detection. Serological detection such as DAS-ELISA has limitations in the production of specific antibodies against virus species from mixed infection (Pramesh et al., 2012). Nucleotidebased detection or molecular techniques such as Reverse Transcription -Polymerase Chain Reaction (RT-PCR) proved more highly specific, reliable and sensitive than ELISA for viral diagnostics (Mohammed et al., 2013;Jones et al., 2017).
In Indonesia, RT-PCR successfully detected the presence of OYDV, SYSV, GarCLV, and SLV in shallot (Swari et al., 2015;Harti et al., 2020;Putri & Hidayat, 2020). Currently, there are not many studies using RT-PCR for detecting virus infection in garlic. Therefore, this study aims to detect OYDV and SLV infecting garlic through RT-PCR using two specific primer pairs. The RT-PCR followed by sequencing was applied to identify viruses infecting garlic in Indonesia accurately. The results of this study provide a method for rapid detection of viruses on garlic and help the government providing virus-free garlic seed bulbs.

Plant Material Preparation
The leaves and bulbs of garlic were collected from several regions such as South Sulawesi (Enrekang), Central Java (Temanggung, Tawangmangu, Magelang), and Yogyakarta.

RNA Extraction
Total RNA was extracted from 0.1 gram leaves or bulbs of garlic. The extraction was carried out according to the protocol from the extraction kit manufacturer (RNesay Plant Mini Kit from Qiagen, Germany). The extraction RNA was used as a template for the synthesis of complementary DNA.

Synthesis of Complementary DNA
Total RNA extracted from the samples subsequently was used for cDNA synthesis. The reactions were performed using ReverTra Ace -α-, cDNA synthesis kit (Toyobo, Japan) with total volume of 10 µl, consisting of 3.5 RNase-free H 2 O; 2 µl 5×RT buffer (contains 25 mM Mg 2+ ); 1 µl dNTPs mixture (10 mM); 0.5 µl primary oligo (dT) 20 (10 pmol/µl); 0.5 µl RNase inhibitor (10 U/µl); 0.5 µl Reverse transcriptase. This mixture was added with 2 µl RNA and homogenized with vortex. The RT-PCR used program of denaturation at 42 o C for 20 minutes and heating 99 o C for 5 minutes to activate the enzyme. The cDNA then used for the template.

PCR Amplification
The DNA amplification reaction of each virus was conducted by PCR method using MyTaqTM HS Red Mix (Bioline, USA) and several primer pairs ( Table 1) that could amplify the virus. The PCR reaction with the total volume of 25 µl, consisted of 12.5 MyTaqTM HS Red Mix, 2 µl of each primer; 6.5 µl ddH 2 O; and 2 µl cDNA templates. Meanwhile, duplex PCR reaction with a total volume of 25 µl, consisted of 12.5 MyTaqTM HS Red Mix, 1 µl of each primer (OYDV and SLV); 6.5 µl ddH 2 O; and 2 µl cDNA templates. The PCR for Potyvirus, Carlavirus, and OYDV, SLV were amplified for 35 cycles: initial denaturation at 95 o C for 3 minutes, denaturation at 95 o C for 1 minute, annealing at temperature (50,51,52,53,54,55,56,57, and 58 o C) for 30 seconds, elongation at 72 o C for 3 minutes, and the final elongation at 72 o C for 10 minutes. The most optimum annealing temperature from all temperatures was 54 o C. Consequently, PCR and duplex PCR used annealing temperature 54 o C for 30 seconds in its PCR amplification. Subsequently, PCR products were electrophoresed in 1% agarose gel, then visualized by UV light. The PCR products were used in nucleotide sequencing. Nurenik et al.:  Nucleotide Sequencing Analysis Analysis of nucleotide sequences was conducted using the sequencer in the Research and Development Center laboratory of PT Genetics Science, Jakarta, Indonesia. Subsequently, the DNA sequence was analyzed by MEGA X and using BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare the target virus sequences with the virus nucleotide sequences from other countries registered in Genbank.

Symptoms in Garlic Plants
Field observation was conducted to discover various symptoms of garlic leaves. The symptoms visual were varied from symptomless, mild, to severe yellow mosaic (Figure 1). The RT-PCR was carried out to assess whether the symptoms were caused by viral agents either OYDV or SLV. Interestingly, those viruses were also detected on symptomless garlic leaves. This might indicate the ability of the plant to tolerate the effects of virus infection or early infection (Jemal et al., 2015). Moreover, yellow mosaic symptoms in garlic plants were also reported elsewhere. Hu et al. (2015) reported the mixed infection of OYDV, SLV, and Allexivirus causing yellow mosaic symptoms in garlic plants was found in China. Meanwhile, in Brazil and Ethiopia, mixed infections of OYDV and Allexivirus have been reported causing yellow mosaic and yellow stripe symptoms on garlic leaves (Jemal et al., 2015;Araújo et al., 2018). Moreover, in West Nusa Tenggara (Indonesia), SLV and GCLV have reported causing yellow mosaic and yellow stripe symptoms in garlic plants (Pauzi et al., 2018). Although all garlic plants presented yellow mosaic symptoms, it was hard to conclude whether all viruses were associated with this symptom or because of the synergistic effect among the viruses. Therefore, in future research, a study for individual viruses in garlic plants was needed to fully understand the effects of each virus on disease symptoms (Takaichi et al., 2001;Da Silva et al., 2019).

Design and Test of Primers Suitable for RT-PCR Detection of Viruses in Garlic
In this study, all targeted fragments were successfully amplified for samples from Enrekang and Tawangmangu. Each primer pair was amplified to produced amplicon at size: 320 bp for Potyvirus using primer MJ1, MJ2, and 715 bp for Carlavirus using primer AlcarF and Poty1 (Figure 2). These universal primer pairs (MJ1, MJ2 and AlcarF, and Poty1) had used by Swari et al. (2015) Table 1. Samples from several regions (Table 2) were used to test the performance of the specific primers designed in this study. Previously all samples were tested using universal primers (data not shown) to ensure that specific primers designed in this study were reliable. All targeted fragments were successfully amplified using the specific primer. Each primer pair amplified an amplicon at size: 112 bp for OYDV and 276 bp for SLV (Figure 3).

Specific Primers for Duplex RT-PCR Detection of Viruses in Garlic
In many countries, duplex and multiplex RT-PCR using specific primers have been successfully used for simultaneous detection of garlic viruses.
In India, duplex RT-PCR using specific primers had successfully detected OYDV and SLV (Majumder et al., 2008). Moreover, multiplex RT-PCR using specific primers was successfully detected OYDV, LYSV, and SLV in China (Hu et al., 2015). In this study, the RT-PCR duplex test of used samples were confirmed with OYDV and SLV infection. Only one sample was successfully amplified from the six samples used. Meanwhile, the other samples only amplified OYDV target, while the SLV target was not amplified (Figure 4). According to Majumder & Baranwal (2014) and Hu et al. (2015), excellent virus-specificity, compatible annealing temperature, and easy to identify amplicon fragment length in gel-electrophoresis are needed to achieve success in duplex or multiplex RT-PCR. This duplex RT-PCR result gave many possibilities include are because different virus concentrations in each sample or

Identification OYDV and SLV from Indonesia
All garlic samples found were infected by OYDV. Meanwhile, double infection (OYDV and SLV) was found in eight out of ten samples (Table 2). Among these two viruses, OYDV was commonly found compared to SLV. Similar conditions were reported in Czech Republic (Leisova-Svobodova & Karlova-Smekalova, 2011), in Indian (Majumder & Baranwal, 2014), and Ethiopia (Abraham et al., 2019), that OYDV was commonly found if compared to other viruses. This indicates that OYDV was abundant in garlic fields in many countries. Mostly garlic farmers in many countries such as China, Ethiopia, and Indonesia, utilized garlic bulbs from seeds that have been previously infected with the viruses from previous season as seeds. Consequently, the virus infection rate in the garlic plants increased (Hu et al., 2015;Jemal et al., 2015). The presence of OYDV and SLV in most garlic plants in this study indicates that those viruses were spread mainly by vegetative propagation.
It was found that garlic viruses distributed from different geographical regions in Indonesia. Such as garlic plants in Bandung and West Nusa Tenggara have been infecting by GCLV and SLV (Kadwati & Hidayat, 2015;Pauzi et al., 2018). Garlic plants in ISSN 1410ISSN -1637, ISSN 2548-4788 (online)  Magelang have been infecting by OYDV. Meanwhile, garlic plants in Enrekang, Temanggung, and Tawangmanggu have been infecting by OYDV and SLV. Due to some garlic production regions have been infecting by various viruses, the prevention of the spreading of those viruses into a new location becomes very important. Hence, the knowledge of the distribution of viruses should be taken into account during the movement trading garlic seed bulbs (Wylie et al., 2014;Bereda et al., 2017). China was the biggest garlic producer in the world and has been exported garlic to various countries including Indonesia (Amanda et al., 2016). If it is traced furthermore, Indonesia has imported garlic for many years from China and isolates OYDV Indonesia has a 95% similar identity with isolate OYDV from China. Therefore, China is probably the source of the OYDV in Indonesia.

CONCLUSION
RT-PCR using specific primers was developed and it successfully detected OYDV and SLV from some regions in Indonesia. Double infection (OYDV and SLV) was commonly found in several regions in Indonesia. This procedure will be useful for the rapid detection of garlic viruses for quarantine applications.