Comparative Cytology of Rice Tungro Viruses in Selected Rice Cultivars

Ultrastructural studies were made using plants of five selected rice cultivars infected with rice tungro viruses. In all cultivars, rice tungro bacilliform virus (RTBV) was found in both xylem and phloem, while rice tungro spherical virus (RTSV) was observed only in the phloem. In tolerant cultivars Balimau Putih and Utri Merah, plants infected with RTBV and RTSV or RTBV alone, had fewer cells containing tungro viruses than plants of the sensitive cultivar Taichung Native 1. In RTBV-sensitive cultivars FK 135 and ASD 8 infected with both viruses or RTBV alone, electron-dense cytoplasm was observed in most cells of the phloem. Abundant phloem necrosis was also observed in FK 135. These observations were correlated with the reaction of each rice cultivar to tungro infection.     

Some biological and genomic properties of rice tungro bacilliform badnavirus and rice tungro spherical waikavirus from Nepal

A survey of rice fields during the main growing seasons in 81 locations from 21 districts of the Southern Terai region of Nepal indicated that rice tungro was primarily restricted to the Hardinath (Janakpur) and Parwanipur (Bara) regions. The tungro incidence in Hardinath ranged from 17% to 51% and in Parwanipur from 6% to 61% causing about 89% grain yield loss in Hardinath. Both rice tungro bacilliform badnavirus (RTBV) and rice tungro spherical picornavirus (RTSV) were found in tungro isolates collected from Hardinath and Parwanipur. These isolates were transmitted by Nephotettix virescens and leaf extracts reacted to antisera against RTBV and RTSV. In a dot blot hybridisation assay, leaf extracts of 12 weed species collected from the tungro-affected area in Hardinath and Parwanipur also reacted with RTBV DNA probes. On mass inoculation of 15 popular rice cultivars most became more than 50% infected and only cv. Radha 9 had low (22.2%) infection. RTBV DNA and the coat protein region of RTSV from the Hardinath isolate were cloned and partially characterised. A comparative analyses by restriction endonuclease digestion, cross hybridisation, the polymerase chain reaction and partial sequencing indicated that the Nepalese RTBV DNA clone and the cDNA clones of the RTSV RNA were more similar to the various tungro isolates from the Indian subcontinent than to those from the Philippines.     

Variation of Rice Tungro Viruses: Further Evidence of Two Rice Tungro Bacilliform Virus Strains and Possibly Several Rice Tungro Spherical Virus Variants

Rice tungro disease is caused by two viruses: rice tungro spherical virus (RTSV) and rice tungro bacilhform virus (RTBV). Our results obtained using polymerase chain reaction (for RTBV) and western blot analysis (for RTSV) to study the epidemiology of tungro supported earlier studies that two RTBV strains. South East Asian and Indian, can be differentiated and also better defined the geographic distribution of these two strains.
Data on RTSV variation were not so conclusive and consistent as those on RTBV because of the high degree of microvariation of RNA genomes. Our approach for differentiation of RTSV led to three variants being identified, the geographic distribution of which does not correlate with that found for strains of RTBV.     

Global Analysis of Rice Tungro Spherical Virus Coat Proteins Reveals New Roles in Evolutionary Consequences

Rice tungro disease is caused by a combination of two viruses: Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). RTSV has a capsid comprising three coat proteins (CP) species. Three CP genes of RTSV-AP isolate were sequenced and compared with 9 other isolates reported worldwide for their phylogenetic survey of recombination events which revealed that in general Indian isolates are forming one separate cluster while those of Philippines and Malaysia forming a different cluster. A significant proportion of recombination sites were found in the CP1 gene, followed by CP2 and CP3 suggesting that it is a major phenomenon in the evolution of various isolates of RTSV. Some interesting domains and motifs such as; 3,4-dihydroxy-2-butanone 4-phosphate synthase in CP1, Type 1 glutamine amidotransferase domain and RNA binding motifs in CP2, domains of receptor proteins in CP3, and glycosylation motif in CP2 and CP3 were also obtained in RTSV coat protein. In addition, simple modular architecture research tool (SMART) analysis of coat proteins of RTSV predicted the coat protein domain of calicivirus suggesting evolutionary linkages between plant and animal viruses. This study provides an opportunity to establish the molecular evolution and sequence-function relationship of RTSV.     

A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors

Rice production is known to be severely affected by virus transmitting rice pests, brown planthopper (BPH) and green leafhopper (GLH) of the order hemiptera, feeding by phloem abstraction. ASAL, a novel lectin from leaves of garlic (Allium sativum) was previously demonstrated to be toxic towards hemipteran pests when administered in artificial diet as well as in ASAL expressing transgenic plants. In this report ASAL was targeted under the control of phloem-specific Agrobacterium rolC and rice sucrose synthase-1 (RSs1) promoters at the insect feeding site into popular rice cultivar, susceptible to hemipteran pests. PCR, Southern blot and C-PRINS analyses of transgenic plants have confirmed stable T-DNA integration and the transgenes were co-segregated among self-fertilized progenies. The T₀ and T₁ plants, harbouring single copy of intact T-DNA expression cassette, exhibit stable expression of ASAL in northern and western blot analyses. ELISA showed that the level of expressed ASAL was as high as 1.01% of total soluble protein. Immunohistofluorescence localization of ASAL depicted the expected expression patterns regulated by each promoter type. In-planta bioassay studies revealed that transgenic ASAL adversely affect survival, growth and population of BPH and GLH. GLH resistant T₁ plants were further evaluated for the incidence of tungro disease, caused by co-infection of GLH vectored Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), which appeared to be dramatically reduced. The result presented here is the first report of such GLH mediated resistance to infection by RTBV/RTSV in ASAL expressing transgenic rice plant.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Deciphering the multiplication behaviour of Rice tungro bacilliform virus by absolute quantitation through real-time PCR

Rice tungro virus disease is one of the most destructive diseases that cause extensive damage to the rice crop. To elucidate the multiplication behaviour of Rice tungro bacilliform virus (RTBV), real-time Polymerase chain reaction (PCR) experiments were performed on rice and insect vector green leafhopper (GLH). SYBR green chemistry-based real-time PCR assay for the quantification of RTBV was developed. A standard curve using plasmid DNA was constructed to determine the absolute quantity of RTBV genome copies in different plant tissues and GLH vector. Here, 6.309?×?10⁴, 7.943?×?10⁵, 3.162?×?10⁶ and 3.162?×?10³ RTBV genome copies per ng of total DNA were estimated in root, shoot, leaf and panicles, respectively, on virus-infected rice cultivar TN1. In addition, 5.011?×?10³ copies of virus in an individual GLH were quantified. Also, RTBV was quantified at different time interval after inoculation. The real-time assay was performed with five different RTBV isolates that showed differential accumulation pattern of virus isolates in a same host. These results provide new insight into the biology of the economically important interaction between rice, GLH and RTBV.     

Inheritance of tolerance to rice tungro bacilliform virus (RTBV) in rice (Oryza sativa L.)

Summary From a large number of rice varieties tested, no variety was identified as resistant to tungro bacilliform virus (RTBV). Only in Utri Merah was the RTBV multiplication restrictive, whereas other varieties such as Kataribhog and Pankhari 203 were identified as tolerant. These varieties were crossed with a susceptible variety. TN1, to study the inheritance of restrictive multiplication and tolerance to RTBV. After 3 weeks of inoculation with RTBV, F₁; F₂, and F₃ progenies were assessed by enzyme-linked immunosorbent assay (ELISA). The RTBV concentration in all F₁ populations was intermediate between parents. The frequency distribution of F₂ seedlings with various levels of RTBV concentration indicated that the RTBV tolerance is controlled by multiple genes. The RTBV concentrations in F₁ and F₂ progenies from the Utri Merah x TN1 cross revealed that restrictive multiplication of RTBV in Utri Merah is a polygenic character. The continuous variation observed in F₂ populations from crosses between tolerant varieties and Utri merah indicated no allelic relationships between tolerant and restrictive multiplication traits.Part of PhD thesis submitted by senior author to University of Kebangsaan Malaysia, Bangi     

Genetic engineering of rice to resist rice tungro disease

Rice tungro disease (RTD), caused by the co-infection of rice tungro bacilliform virus (RTBV) and rice tungro spherical virus, is one of the most important viral diseases of rice in South and Southeast Asia. The disease remains one of the major threats to sustainable rice production in many countries. The lack of resistance genes to RTBV—the causal agent of tungro disease—makes it even more difficult to manage RTD. In this review, we summarize previous and current research efforts to genetically engineer rice in order to increase the crop’s resistance to tungro disease, including the use of pathogen-derived resistance and of host genes that confer RTD resistance and/or that restrict feeding by the insect vector. The prospects of developing rice cultivars with durable resistance to RTD are also discussed.     

The Differential Reaction of Rice Hybrids to Tungro Virus by Phenotyping and PCR Analysis

Twenty popular rice hybrids were used to screen for rice tungro virus (RTV) disease reaction. Virulent green leafhoppers (GLH) were used as vector to introduce RTV to the rice hybrids. Virus symptoms scores were recorded at 14, 21, 34, 41 and 59 days postinoculation (DPI), which suggested that virus symptoms are greatly influenced by growth stage of plants. To confirm the presence of virus, polymerase chain reaction (PCR)‐based detection of Rice tungro bacilliform virus (RTBV) was carried out at 7, 14, 21 and 59 DPI using virus genome‐specific primers. Virus presence was observed in all the rice hybrids and check varieties, particularly at later stages of infection. This study shows that phenotyping for tungro virus resistance in rice hybrids at 21 DPI gives most reliable results based on both virus symptoms and presence of virus. Further, to assess the relative difference in population of RTBV, quantitative PCR was performed in all the genotypes at 21 DPI. Yield data were also recorded from control and virus‐infected plants to estimate yield loss percentage due to tungro disease. This study is important to understand the response of rice hybrids to tungro virus disease. Results obtained in this study emphasize that molecular detection of virus is very important to screen the rice plants accurately for tungro disease reaction.     

The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.     

Genetic Analysis of Tolerance to Rice Tungro Bacilliform Virus in Rice (Oryza sativa L.) Through Agroinoculation

Balimau Putih [an Indonesian cultivar tolerant to rice tungro bacilliform virus (RTBV)] was crossed with IR64 (RTBV, susceptible variety) to produce the three filial generations F₁, F₂ and F₃. Agroinoculation was used to introduce RTBV into the test plants. RTBV tolerance was based on the RTBV level in plants by analysis of coat protein using enzyme‐linked immunosorbent assay. The level of RTBV in cv. Balimau Putih was significantly lower than that of IR64 and the susceptible control, Taichung Native 1. Mean RTBV levels of the F₁, F₂ and F₃ populations were comparable with one another and with the average of the parents. Results indicate that there was no dominance and an additive gene action may control the expression of tolerance to RTBV. Tolerance based on the level of RTBV coat protein was highly heritable (0.67) as estimated using the mean values of F₃ lines, suggesting that selection for tolerance to RTBV can be performed in the early selfing generations using the technique employed in this study. The RTBV level had a negative correlation with plant height, but positive relationship with disease index value.     

Genome editing of indica rice ASD16 for imparting resistance against rice tungro disease

Journal of Plant Biochemistry and Biotechnology - Rice tungro disease (RTD) is caused by the joint infection of rice tungro bacilliform virus and rice tungro spherical virus (RTSV) and is the most...     

Interaction Between Root and Leaf Disease Development in Barley Cultivars after Inoculation with Different Isolates of Bipolaris sorokiniana

The relationship between root and leaf infection in 11 cultivars of barley ( Hordeum vulgare ) by different isolates of Bipolaris sorokiniana was investigated in young plants. Roots of 10-day-old seedlings, grown in filterpaper rolls, and the third leaf of 17-day-old seedlings were inoculated with the different isolates and a Disease Development Index (DDI) was calculated.
The rate of lesion development in leaves was higher than in roots, indicated by generally higher DDI after leaf inoculation than after root inoculation. Significant differences in resistance were found among the barley cultivars. Inoculation with different isolates of B. sorokiniana caused significant differences in DDI for both roots and leaves. In the leaves, but not in the roots, a significant cultivar–isolate interaction was found. No significant correlations, neither in isolate aggressiveness nor in cultivar reaction between root and leaf, were observed.     

Genetic control of rice blast resistance in the durably resistant cultivar Gumei 2 against multiple isolates

To further our understanding of the genetic control of blast resistance in rice cultivar Gumei 2 and, consequently, to facilitate the utilization of this durably blast-resistant cultivar, we studied 304 recombinant inbred lines of indica rice cross Zhong 156/Gumei 2 and a linkage map comprising 181 markers. An analysis of segregation for resistance against five isolates of rice blast suggested that one gene cluster and three additional major genes that are independently inherited are responsible for the complete resistance of Gumei 2. The gene cluster was located to chromosome 6 and includes two genes mapped previously, Pi25(t), against Chinese rice blast isolate 92-183 (race ZC15) and Pi26(t) against Philippine rice blast isolate Ca89 (lineage 4), and a gene for resistance against Philippine rice blast isolate 92330-5 (lineage 17). Of the two genes conferring resistance against the Philippine isolates V86013 (lineage 15) and C923-39 (lineage 46), we identified one as Pi26(t) and mapped the other onto the distal end of chromosome 2 where Pib is located. We used three components of partial blast resistance, percentage diseased leaf area (DLA), lesion number and lesion size, all measured in the greenhouse, to measure the degree of susceptibility to isolates Ca89 and C923-39 and subsequently identified nine and eight quantitative trait loci (QTLs), respectively. Epistasis was determined to play an important role in partial resistance against Ca89. Using DLA measured on lines susceptible in a blast nursery, we detected six QTLs. While different QTLs were detected for partial resistance to Ca89 and C923-39, respectively, most were involved in the partial resistance in the field. Our results suggest that the blast resistance in Gumei 2 is controlled by multiple major genes and minor genes with epistatic effects.     

The Response of Lettuce Cultivars against Two Lettuce mosaic virus isolates,One Able to Systemically Infect the Resistant Cultivar Salinas 88

The response of seven lettuce cultivars to two geographically different Lettuce mosaic virus (LMV) isolates (LMV‐A, LMV‐T) was statistically evaluated based on infection rate, virus accumulation and symptom severity in different time trials. LMV‐A is characterized by the ability to systemically infect cv. Salinas 88 (mo1²‐carrying resistant cultivar), and inducing mild mosaic symptoms. Among lettuce cultivars, Varamin (a native cultivar) similar to cv. Salinas showed the most susceptibility to both LMV isolates, whereas another native cultivar, Varesh, was tolerant to the virus with minimal viral accumulation and symptom scores, significantly different from other cultivars at P < 0.05. LMV‐A systemically infects all susceptible lettuce cultivars more rapidly and at a higher rate than LMV‐T. This isolate accumulated in lettuce cultivars at a significantly higher level, determined by semiquantitative ELISA and induced more severe symptoms than LMV‐T isolate at 21 dpi. This is the first evidence for a LMV isolate with ability to systemically infect mo1²‐carrying resistant cultivar of lettuce from Iran. In this study, accumulation level of LMV showed statistically meaningful positive correlation with symptom severity on lettuce plants. Based on the results, three evaluated parameters differed considerably by lettuce cultivar and virus isolate.