Sequence and phylogenetic analysis of the RNA1 and RNA2 segments of Korean Rice stripe virus isolates and comparison with those of China and Japan

Jonson, Miranda Gilda,Choi, Hong-Soo,Kim, Jeong-Soo,Choi, Il-Ryong,Kim, Kook-Hyung Springer-Verlag 2009 Archives of virology Vol.154 No.10

Inoculum Sources to Generate High Mechanical Transmission of Barley yellowmosaic virus

Gilda Jonson,Yang-Kil Kim,Mi-Jung Kim,Jong-Chul Park,Jong-Nae Hyun,Jung-Gon Kim 한국식물병리학회 2007 Plant Pathology Journal Vol.23 No.2

Mechanical transmission of barley seedlings with barley yellow mosaic virus (BaYMV) is generally inefficient and is the major constraint for testing cultivar resistance to the virus. To explore mechanical transmission, BaYMV-infected barley plants were grown at different conditions and used as inoculum sources to seedlings of susceptible barley cultivar Baegdong. Extracts prepared from BaYMV-infected Baegdong plants at 47, 53, 74, and 90 days after symptom appearance (DASA) and grown at 10 and 12ºC gave 10, 30, 68 and 76% infection, respectively on inoculated susceptible barley cv. Baegdong seedlings. While Jinyangbori, another susceptible cultivar obtained 95% infection rate inoculated with extracts from 90 DASA disease source and grown at 10/12ºC. However, low infection rates were obtained when the virus sources were grown in a greenhouse at 15-18ºC. Our results indicate that longer incubation period and lower temperature are required for virus accumulation and stability.

Direct Stem Blot Immunoassay (DSBIA):

Gilda Jonson,박종철,김상길,김미정,이미자,현종내,김정군 한국식물병리학회 2007 Plant Pathology Journal Vol.23 No.4

Testing a large number of samples from field monitoring and routine indexing is cumbersome and the available virus detection tools were labor intensive and expensive. To circumvent these problems we established tissue blot immunoassay (TBIA) method an alternative detection tool to detect Barley mild mosaic virus (BaMMV) and Barley yellow mosaic virus (BaYMV) infection in the field and greenhouse inoculated plants for monitoring and routine indexing applications, respectively. Initially, leaf and stem were tested to determine suitable plant tissue for direct blotting on nitrocellulose membrane. The dilutions of antibodies were optimized for more efficient and economical purposes. Results showed that stem tissue was more suitable for direct blotting for it had no background that interferes in the reaction. Therefore, this technique was referred as direct stem blot immunoassay or DSBIA, in this study. Re-used diluted (1:1000) antiserum and conjugate up to 3 times with the addition of half strength amount of concentrated antibodies was more effective in detecting the virus. The virus blotted on the nitrocellulose membrane from stem tissues kept at room temperature for 3 days were still detectable. The efficiency of DSBIA and RT-PCR in detecting BaMMV and BaYMV were relatively comparable. Results further proved that DSBIA is a rapid, reliable and economical detection method suitable for monitoring BaMMV and BaYMV infection in the field and practical method in indexing large scale of barley materials for virus resistance screening.

Development of an Efficient Mechanical Inoculation Technique to Screen Barley Genotypes for Resistance to Barley mild mosaic virus Disease and its Comparison to Natural Infection

Gilda Jonson,Jong-Chul Park,Tae Hwan Noh,Mi-Jung Kim,Jong-Nae Hyun,Jung-Gon Kim 한국식물병리학회 2006 Plant Pathology Journal Vol.22 No.4

Bymovirus vectored by root-infecting fungus, Polymyxa graminis. Mechanism of cultivar’s resistance to BaMMV in field tests are difficult to assess since resistance could be either due to the virus or to P. graminis, or both. Whereas, available mechanical inoculation methods for BaMMV and other related viruses are labor intensive, give inconsistent results and generally result in low infection rates. Inoculation method using stick with gauze (SWG) was developed for BaMMV. The improved method proved to be simple, efficient, and reliable. The infected leaf tissues were preserved by drying in a frozen state under high vaccum (freeze dried barley infected leaves) to circumvent reduction of virus infectivity during storage. Five Korean barley cultivars were mechanically inoculated with BaMMV-infected sap by the improved method. Infection rates obtained were compared with natural infection. Cultivar Naehanssalbori showed resistance to BaMMV in the field trials but was found highly susceptible in the greenhouse tests by mechanical inoculation, indicating that the field resistance may be possibly due to resistance to P. graminis.

Effects of Recombination on the Pathogenicity and Evolution of Pepper mottle virus

Jonson, Miranda Gilda,Seo, Jang-Kyun,Cho, Hong-Soo,Kim, Jeong-Soo,Kim, Kook-Hyung The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.4

The analysis of the full length genome of Korean isolates of Pepper mottle virus (PepMoV) in previous study showed molecular variations and are found to be related to symptom variation and pathogenicity (Kim et al., 2009, Virus Res. 144:83-88). To fully understand the molecular variation of PepMoV in Korea, we further assessed the role of RNA recombination to biological variation and evolution of PepMoV. Full-length genome of a total of 17 Korean-PepMoV and 2 American (CA and FL) isolates were examined for possible detection of genetic recombination using different recombination detections programs and detected 5 and 8 tentative recombination events using RDP3 and Splits Tree4 programs, respectively. Interestingly, tentative recombinants detected such as isolates 57, 134 and 217 were previously identified as severe isolates and 205135 and 205136 as differentiating isolates (Kim et al., 2009, Virus Res. 144:83-88). In addition, recombination was frequently detected in the Vb isolate, the first PepMoV isolate reported in Korea, suggesting significant involvement in the evolution of PepMoV in Korea. These initial results of our recombination analyses among PepMoV isolates in Korea may serve as clues to further investigate the biological variations and evolution of PepMoV brought about by recombination.

Complete Genome Sequence of the RNAs 3 and 4 Segments of Rice stripe virus Isolates in Korea and their Phylogenetic Relationships with Japan and China Isolates

Jonson, Miranda Gilda,Choi, Hong-Soo,Kim, Jeong-Soo,Choi, Il-Ryong,Kim, Kook-Hyung The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.2

The complete genome sequences of RNA3 and RNA4 of the 13 different Rice stripe virus (RSV) isolates were determined and characterized in this study to address the possible causes of the recent re-emergence of RSV that affected many rice fields in Korea. The genome size of each RNA segment varied among isolates and significant differences were observed in the intergenic region. There was up to 4% average divergence in the RNA4 nucleotide sequence among 13 Korean isolates and only 1.4% in the RNA3. Phylogenetic relationships among different Korean isolates revealed that there were at least 2 types of RNA3 and 4 distinct types of RNA4 genomes present in Korea. However, Korean isolates with one type of RNA3 predominate over the other while the occurrences of the RSV Korean isolates with the 4 types of RNA4 genome were not correlated to specific geographical areas. Results further indicate that RNA4 had diverged more than RNA3 and these differences in accumulation of mutations in the individual RNA segments indicate that genetic reassortment were likely to contribute to the genetic divergence in the 13 Korean isolates. All of the Korean-RNA3 sequences except for one isolate grouped with Chinese isolates (JY and Z). In contrast, the RNA 4 sequences segregated together with either Chinese (JY and Z) and Japanese (M and T) isolates but genetic relationships of Korean isolates- RNAs 3 and 4 segments to Chinese-Y isolate were low. Altogether, these results suggest that the occurrence of mixtures of RNAs 3 and 4 genotypes in the natural population of RSV may have contributed to the sudden outbreak in Korea.

Genetic Reassortment of Rice stripe virus RNA Segments Detected by RT-PCR Restriction Enzyme Analysis-based Method

Jonson, Miranda Gilda,Lian, Sen,Choi, Hong-Soo,Lee, Gwan-Seok,Kim, Chang-Suk,Kim, Kook-Hyung The Korean Society of Plant Pathology 2011 Plant Pathology Journal Vol.27 No.2

Our previous sequence and phylogenetic analyses of the Korean Rice stripe virus (RSV) suggested possible genetic reassortment of RNA segments, but whether this RNA variation contributed to the recent RSV outbreaks in Korea is yet unclear. To further clarify these RSV-RNA segment variations, we developed a reverse transcription-polymerase reaction/restriction enzyme (RT-PCR/RE) analysis-based method. We identified five REs, including DraI, EcoR1, NdeI/AseI, and SpeI, that could differentiate RSV RNA 1-4 subtypes, respectively. Our RT-PCR/RE results provided a clear pattern of RNA reassortment, i.e., different groups of isolates having their RNA segments derived from two to three different RSV ancestors, such as from Eastern and Southwestern Chinese or Japanese M and T isolates. We also found that the migratory small brown planthopper from Eastern China caught by aerial net traps that possesses RSV-RNA3 genotypes corresponds mainly to Eastern China, with a few for Southwestern China based on RT-PCR/RE, sequence and phylogenetic analyses, indicating that RSV populations in Eastern China may also have strong RNA variation. The development of an RE analysisbased method proved a useful epidemiological tool for rapid genotyping and identification of mixed infections by RSV strain and by different subtype.

Development of an Efficient Mechanical Inoculation Technique to Screen Barley Genotypes for Resistance to Barley mild mosaic virus Disease and its Comparison to Natural Infection

Jonson, Gilda,Park, Jong-Chul,Noh, Tae-Hwan,Kim, Mi-Jung,Hyun, Jong-Nae,Kim, Jong-Gon The Korean Society of Plant Pathology 2006 Plant Pathology Journal Vol.22 No.4

Barley mild mosaic virus(BaMMV) is a soilborne Bymovirus vectored by root-infecting fungus, Polymyxa graminis. Mechanism of cultivar's resistance to BaMMV in field tests are difficult to assess since resistance could be either due to the virus or to P. graminis, or both. Whereas, available mechanical inoculation methods for BaMMV and other related viruses are labor intensive, give inconsistent results and generally result in low infection rates. Inoculation method using stick with gauze(SWG) was developed for BaMMV. The improved method proved to be simple, efficient, and reliable. The infected leaf tissues were preserved by drying in a frozen state under high vaccum(freeze dried barley infected leaves) to circumvent reduction of virus infectivity during storage. Five Korean barley cultivars were mechanically inoculated with BaMMV-infected sap by the improved method. Infection rates obtained were compared with natural infection. Cultivar Naehanssalbori showed resistance to BaMMV in the field trials but was found highly susceptible in the greenhouse tests by mechanical inoculation, indicating that the field resistance may be possibly due to resistance to P. graminis.

Inoculum Sources to Generate High Mechanical Transmission of Barley yellow mosaic virus

Jonson, Gilda,Kim, Yang-Kil,Kim, Mi-Jung,Park, Jong-Chul,Hyun, Jong-Nae,Kim, Jung-Gon The Korean Society of Plant Pathology 2007 Plant Pathology Journal Vol.23 No.2

Mechanical transmission of barley seedlings with barley yellow mosaic virus (BaYMV) is generally inefficient and is the major constraint for testing cultivar resistance to the virus. To explore mechanical transmission, BaYMV-infected barley plants were grown at different conditions and used as inoculum sources to seedlings of susceptible barley cultivar Baegdong. Extracts prepared from BaYMV-infected Baegdong plants at 47, 53, 74, and 90 days after symptom appearance (DASA) and grown at 10 and $12^{\circ}C$ gave 10, 30, 68 and 76% infection, respectively on inoculated susceptible barley cv. Baegdong seedlings. While Jinyangbori, another susceptible cultivar obtained 95% infection rate inoculated with extracts from 90 DASA disease source and grown at $10/12^{\circ}C$. However, low infection rates were obtained when the virus sources were grown in a greenhouse at $15-18^{\circ}C$. Our results indicate that longer incubation period and lower temperature are required for virus accumulation and stability.

Direct Stem Blot Immunoassay (DSBIA): A Rapid, Reliable and Economical Detection Technique Suitable for Testing Large Number of Barley Materials for Field Monitoring and Resistance Screening to Barley mild mosaic virus and Barley yellow mosaic virus

Jonson, Gilda,Park, Jong-Chul,Kim, Yang-Kil,Kim, Mi-Jung,Lee, Mi-Ja,Hyun, Jong-Nae,Kim, Jung-Gon The Korean Society of Plant Pathology 2007 Plant Pathology Journal Vol.23 No.4

Testing a large number of samples from field monitoring and routine indexing is cumbersome and the available virus detection tools were labor intensive and expensive. To circumvent these problems we established tissue blot immunoassay (TBIA) method an alternative detection tool to detect Barley mild mosaic virus (BaMMV) and Barley yellow mosaic virus (BaYMV) infection in the field and greenhouse inoculated plants for monitoring and routine indexing applications, respectively. Initially, leaf and stem were tested to determine suitable plant tissue for direct blotting on nitrocellulose membrane. The dilutions of antibodies were optimized for more efficient and economical purposes. Results showed that stem tissue was more suitable for direct blotting for it had no background that interferes in the reaction. Therefore, this technique was referred as direct stem blot immunoassay or DSBIA, in this study. Re-used diluted (1:1000) antiserum and conjugate up to 3 times with the addition of half strength amount of concentrated antibodies was more effective in detecting the virus. The virus blotted on the nitrocellulose membrane from stem tissues kept at room temperature for 3 days were still detectable. The efficiency of DSBIA and RT-PCR in detecting BaMMV and BaYMV were relatively comparable. Results further proved that DSBIA is a rapid, reliable and economical detection method suitable for monitoring BaMMV and BaYMV infection in the field and practical method in indexing large scale of barley materials for virus resistance screening.