Molecular Detections and Resistance Response of Six Rice Varieties to Tungroviruses from South Sulawesi

South Sulawesi is the fourth highest rice production center in Indonesia of 5.74 million tons (BPS, 2018). One of the difficulties in rice production is the tungrovirus. Tungro disease has been reported in Sidrap, South Sulawesi, in 2018 covering an area of 6 ha, and now there are many symptoms similar to tungro disease. The most common symptoms of tungro are dwarf, yellowish leaves, stunted growth, and the inability to produce panicle. Tungro disease can reduce rice production and even cause puso (harvest failure) if the infection occurs since the beginning of the vegetative phase or at the nursery stage (Hasanuddin, 2002). Tungro is caused by two different types of viruses: stem-shaped virus, Rice tungro bacilliform virus (RTBV) with DNA type genome; and spherical virus, Rice tungro spherical virus (RTSV) with RNA type. RTBV has a diameter of 35 × 150–350 nm with a length of 100.300 nm while the RTSV has a diameter of 30 nm (Hibino et al., 1978; Omura et al., 1983). Both types of viruses do not have the same serological kinship but can infect plants together without causing cross-protection between the viruses (Mukhopadhyay, 1995). The tungrovirus is only transmitted by green leafhoppers in a semi-persistently (Hibino & Cabunagan, 1986). Molecular detection with Polymerase Chain Reaction (PCR) techniques to detect viruses with DNA genome and Reverse Transcription (RT)-PCR for viruses with RNA genome is very sensitive and accurate compared to other methods such as serology and nucleic acid hybridization. (Takahashi et al., 1993). The PCR technique is very advantageous in detecting the presence of rice viruses because it is easier and faster than other techniques such as in South Sulawesi, one of the largest rice production centers in Indonesia, which currently has many tungro disease symptoms. The symptoms of the outbreak are varied and the intensity is getting higher hence ABSTRACT


INTRODUCTION
South Sulawesi is the fourth highest rice production center in Indonesia of 5.74 million tons (BPS, 2018). One of the difficulties in rice production is the tungrovirus. Tungro disease has been reported in Sidrap, South Sulawesi, in 2018 covering an area of 6 ha, and now there are many symptoms similar to tungro disease. The most common symptoms of tungro are dwarf, yellowish leaves, stunted growth, and the inability to produce panicle. Tungro disease can reduce rice production and even cause puso (harvest failure) if the infection occurs since the beginning of the vegetative phase or at the nursery stage (Hasanuddin, 2002).
Tungro is caused by two different types of viruses: stem-shaped virus, Rice tungro bacilliform virus (RTBV) with DNA type genome; and spherical virus, Rice tungro spherical virus (RTSV) with RNA type. RTBV has a diameter of 35 × 150-350 nm with a length of 100.300 nm while the RTSV has a diameter of 30 nm (Hibino et al., 1978;Omura et al., 1983). Both types of viruses do not have the same serological kinship but can infect plants together without causing cross-protection between the viruses (Mukhopadhyay, 1995). The tungrovirus is only transmitted by green leafhoppers in a semi-persistently (Hibino & Cabunagan, 1986).
Molecular detection with Polymerase Chain Reaction (PCR) techniques to detect viruses with DNA genome and Reverse Transcription (RT)-PCR for viruses with RNA genome is very sensitive and accurate compared to other methods such as serology and nucleic acid hybridization. (Takahashi et al., 1993). The PCR technique is very advantageous in detecting the presence of rice viruses because it is easier and faster than other techniques such as in South Sulawesi, one of the largest rice production centers in Indonesia, which currently has many tungro disease symptoms. The symptoms of the outbreak are varied and the intensity is getting higher hence apart from being based on the symptoms it is necessary to further identify the distribution and cause so that it can be used as a basis for developing effective and environmental-friendly control strategies.
Many methods are used to control tungro disease such as the use of insecticides to control planthopper. However, this method is considered less effective and harms the environment. One ofthe environmentalfriendly control alternatives is using varieties resistant to the tungrovirus and green leafhopper as vector insects (Sama, 1985cit. Praptana & Muliadi, 2005.According to Suprihatno (1985) cit. , known and utilized sources of tungro disease resistance genes are Latisail, CR-94-13, Gam-Pai 15, and resistant varieties which are crossed breeding from those parents. Varieties with vertical resistance have always been a mainstay in reducing the planthopper. The use of resistant varieties is constrained by the adaptability of green leafhoppers by forming new biotypes so that the varieties that are released shortly afterward was broken their resistance. Tungro disease infection in resistant varieties causes no symptoms in the form of a slightly yellowish leaf that disappears as the plant ages (Choi et al., 2009). Tungro symptoms would begin to appear when the plants aged 10-15 days after the inoculation of the virus, whereas in fields, symptoms would appear when the plants are 21-30 days after planting (Raga et al., 2004). This study aimed to detect the presence of the tungrovirus molecularly in South Sulawesi and determine the response of the resistance of some rice varieties to the tungrovirus.

MATERIALS AND METHODS
The survey was conducted in several districts of rice production centers in

RTSV Detection Using RT-PCR Technique
The detection phase of RTSV began with the extraction of total RNA from rice leaf samples, the stage of total RNA extraction followed the protocol of the commercial kit (mini RNA kit plant, Geneaid). The total RNA extracted was used as a template in the reverse transcription reaction to produce cDNA (complementary DNA). The making of cDNA was performed by the RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) method with a total volume of 10 µl containing 2 µl total RNA of, 3.5 µl RNase Free H 2 O, 0.5 µl RNase Inhibitor, 0.5 µl ReverTraAcc, 2 µl 5x RT Buffer, 1 µl dNTP Mixture, and 0.5 µl oligo primer (dt) 20. The reverse transcription reaction was carried out at 42ºC for 20 minutes, followed at 99ºC for 5 minutes, and at last 4ºC. The result of cDNA was used as a DNA template in the amplification reaction.
Amplification was conducted using a specific pair of RTSV-F primers (AAACGGTCATTGTGG GGAGGT) and RTSV-R (CAGGCCCAGCAACG ACATAA) with a target of 1115 bp (Shenet al., 1993). The reaction for PCR was made with a total volume of 10 µl containing 0.5 µl RTSV-F primers, 0.5 µl RTSV-R primers, 3 µl DH 2 O, 1 µl cDNA Samples, and 5 µl MyTaqTM HS RedMix PCR Mix. The amplification process was preceded by initial denaturation temperature of 95ºC for 2 minutes, denaturation temperature of 95ºC for 30 seconds, the annealing temperature of 57ºC for 30 seconds, extension temperature of 72ºC for 30 seconds, the final temperature of extension of 72ºC for 7 minutes, and the hold temperature of 4ºC ∞, over35 cycles. The amplification results were electrophoresed at 100 V for 30 minutes and colored with ethidium bromide (0.5 g/ml) for 15 minutes. The results of DNA visualization on the UV transilluminator were then documented with a digital camera.

RTBV Detection Using PCR Technique
RTBV detection began with the extraction of total DNA from each rice leaf sample. Total DNA extraction followed the protocol of a commercial kit (DNA Mini kit plant, Geneaid). The total DNA was used as a DNA template in the PCR reaction using a pair of RTBV-B2F specific primers (GCAGAA CAGAACTCTAAGGC) and RTBV-B2R (GTCTAA GGCTCATGCTGGAT) with product target of 430 bp (Cabauatan et al., 1999). The PCR reaction was made with a total volume of 10 µl containing 1 µl DNA template, 0.5 µl RTBV-B2F primer, 0.5 µl RTBV-B2R primer, 3 µl DH 2 O, and 5 µl MyTaqTM HS RedMix PCR Mix. The amplification process was performed over 35 cycles preceded by initial denaturation temperature of 95ºC for 2 minutes, denaturation temperature of 95ºC for 30 seconds, the annealing temperature of 53ºC for 30 seconds, temperature extension 72ºC for 30 seconds, final extension temperature of 72ºC for 7 minutes, and the hold temperature at 4ºC ∞. The amplification results were electrophoresed at 100 V for 30 minutes and colored with ethidium bromide (0.5 g/ml) for 15 minutes. Visualization of the amplified DNA results was similaras described previously.

Tungrovirus Transmission Test in Six Rice Varieties
Samples of rice contained both types of tungrovirus (RTSV and RTBV) based on the results of detection by PCR then used as a source of inoculum. The acquisition of the virus was carried out by inoculating 200 green leafhoppers into confinement containing rice plants that were positively infected by the two tungroviruses over 24 hours for the acquisition feeding period. The inoculation feeding by green leafhoppers was conducted for 24 hours on healthy rice seeds of each variety aged 1 week using the test tube method (tube test). Each test tube (in a total of 10 tubes) containing 1 plant and 1 green leafhopper with 3 replications. Inoculated seedlings were then transplanted in pots containing planting media. Observation of symptoms was done after the plants aged 2 weeks and scoring based on the Standard Evaluation System for Rice (IRRI, 1996). The formula for Disease Incidence (I) and Disease Intensity (DI) (Zadoks & Schein, 1979) are as follows:

RESULTS AND DISCUSSION
Observation tungro disease symptoms on rice were carried out on the rice fields owned by farmers in several districts in South Sulawesi consisting of 19 observation locations and 4 districts (Wajo, Maros, Pinrang, and Sidrap). Several symptoms of tungro disease were found, i.e. yellowish leaf, twisting, stunted, and increasedtillers, with the disease incidence ranged of 10-55% (Table 3). It was suspected that the virus was transmitted by green leafhoppers in the previous planting season and then survived on weeds and the rest of the rice plants that have been harvested (ratun) around the rice fields. The disease intensity in the fields in Kabrinang was 5-10%, Maros Regency was 5-10%, Sidrap was 5-15%, and Wajowas 5-12%. Insect populationvector such as green leafhoppers also contributed to the growth of rice plants in the field, while observations on fields in several districts in South Sulawesi showed that the tungrovirus attack had a different disease intensity and insect population vector. The higher the number of insect populations vector, the higher the disease incidence caused, as in Sidrap has the highest disease incidence of 55% with a population of 10 insect vectors (Table 3).
The most likely symptom detected in the field was leaf discoloration and differences in the plant height (uneven growth) from visual observation. Tungro and stunted symptomatic plants were found clustered in one plot and there were uneven spots and plant growths seen on the rice fields. Vectors play an important role in the transmission and spread of the viruses. The highest population of green leafhopper obtained from Sidrap and Maros, ranged from 10-14 individuals, with disease incidence reached 50-55% (Table 3) showed that the higher the vector population density, the higher the disease incidence (Hibino & Cabunagan, 1986).

Tungrovirus Detection by PCR
PCR analysis results indicated that the presence of rice tungrovirus had been detected, namely RTBV and RTSV on plant samples obtained in several districts in South Sulawesi. Observation of disease incidence in the field was found with severe symptoms of tungro ISSN 1410-1637 (print), ISSN 2548-4788 (online)  (Figure 1), this was proven by laboratory test results using PCR techniques showed that the Pinrang sample had been infected by RTSV with DNA band size of 1115 bp and RTBV of 430 bp (Figure 2). Other samples such as Sidrap and Maros isolates positively infected by RTBV, based on observations in the field with mild symptoms and also found a green leafhopper vector. Detection results indicated that the tungrovirus infection had transmitted by green leafhopper.

Sequencing Analysis
Homology analysis of RTBV showed that the first subgroup (Sidrap, Maros, and Pinrang) had a kinship of 97%. Meanwhile, the second subgroup (Philippines IC/G1) had a kinship of 94% and the third subgroup (Chainat-Thailand, Seberang Perai-Malaysia, and Serdang-Malaysia) of 92% (Table 4). The results of the dendrogram analysis showed that the sample from Sidrap had a very close relationship and belonged to one group with the sample from Maros and Pinrang, but had a close or different group from the Philippines, Malaysia and Thailand samples (Figure 3).
RTSV homology analysis showed that the RTSV nucleotide sequences of Pinrang had similarities between nucleotide bases and Subang (92.49%), Bali (92.69%), India (82.96%), Malaysia (84.38%), and Philippines (85.80%)( Table 5). This finding indicated that the RTSV sample in Indonesia had a close relationship of 92% compared to samples from other Asian regions, such as India, Malaysia, and the Philippines ranging from 82-85% (Table 5). This was similar to King et al. (2012) that a virus has a close kinship if it has a homology of nucleotide sequences >89%. Dendrogram of the genetic relationship between RTSV samples based on the nucleotide base sequence of polyprotein gene sequences showed that the Pinrang sample was in one group with AF113827-Subang and AF113823-Bali samples. (Figure 4). The variations in the nucleotide bases that make up the DNA and amino acid sequences produce the genetic diversity of RTSV samples. This is because the diversity of the nucleotide structure and virulence rate of the tungrovirus in Southeast Asia differ from the tungrovirus gene in South Asia (Azzam & Chancellor, 2002).

The Response of Six Rice Varieties to Tungrovirus
Based on theresistance test of six rice varieties showed that there were symptoms and different plant heights were observed, namely yellowish of leaves from the tips to the base ( Figure 5). Disease Incidence (I) ranged from 40-100% with the highest Icame from TN1 variety (100%) and the lowest was Inpari 36 (40%). While the highest Disease Intensity (DI) was TN1 variety (60.66%, susceptible) and the lowest was Inpari 36 (22.21%, resistant). The average incidence of tungro disease from transmission in each variety ranged from 40-100%, with an average incubation period of 8-17 days, while the intensity of tungro disease ranged from 22-60% (Table 7).
All varieties inoculated with tungrovirus showed symptoms of the tungro diseasse. TN1 and Ciherang varieties showed the most severe symptoms until the plant became stunted and had a yellowish color.     , some varieties that are initially known to be resistant in one location would show a susceptible reaction in other locations, and previously susceptible varieties became resistance in other locations. The resistance of rice varieties to green leafhopper vectors is also determined by other factors, i.e. biochemical factors such as nutrient content and biophysical factors (plant tissue thickness or the interaction of these two factors on reproductive cells) hence it affects the number and hatching rate of green leafhopper eggs (Pakki, 2011). Several varieties of rice tested showed that not all plants could be infected by the tungrovirus. The severity score of disease symptoms per plant was mostly 1, 3, and 5, and only a few plants were valued 7 and 9 (Table 6). Furthermore, there were differences in disease intensity between resistant, slightly resistant, and susceptible varieties (Table 7). Tungro disease symptoms were characterized by leaf discoloration from green to yellow-orange and stunted growth about 1-10% compared that to the control plants of each variety. Each variety could potentially be infected by the tungrovirus. The intensity of the disease with the same or different values depends on the variety planted. This finding showed that there were different varieties in responding to the tungrovirus infection by green leafhoppers.

CONCLUSION
Viruses detected using the PCR technique was Rice tungro bacilliform virus (RTBV) DNA band size of 430 bp from Maros, Sidrap, and Pinrang samples; and Rice tungro spherical virus (RTSV) DNA band size of 1115 bp from Pinrang sample. Transmission of the tungrovirus in the greenhouse showed that the response of resistance to six different rice varieties in terms of disease intensity, disease incidence, and incubation period. The highest percentage of tungro disease intensity was from TN1 variety, while the slightly resistant rice varieties were Mekongga and Tukad Unda, and resistantvarieties were Inpari 36 and Inpari 37.