The Biology, Epidemiology, and Management of Rice Tungro Disease in Asia

Many farmers in South and Southeast Asia describe rice tungro disease as a cancer disease because of the severe damage it causes and the difficulty of controlling it (121). As the most important of the 14 rice viral diseases, tungro was first recognized as a leafhopper-transmitted virus disease in 1963 (88). However, tungro, which means “degenerated growth” in a Filipino dialect, has a much longer history. It is almost certain that tungro was responsible for a disease outbreak that occurred in 1859 in Indonesia, which was referred to at the time as mentek (83). In the past, a variety of names has been given to tungro, including accep na pula in the Philippines, penyakit merah in Malaysia, and yelloworange leaf in Thailand (83).

Synchrony of planting and proportions of susceptible varieties affect rice tungro disease epidemics in the Philippines.

Surveys were conducted in the Philippines from 1995 to 1997 to examine relationships between production environment variables (agroecosystem, synchrony of planting, and varieties planted) and the occurrence of rice tungro disease epidemics using correspondence analyses. The sites covered were Isabela, Nueva Ecija, North Cotabato, and Bohol provinces as well as Bicol region. Tungro disease incidence in farmers’ fields was assessed visually based on typical symptoms. In addition, leaf samples were collected from each field and indexed serologically by enzyme-linked immunosorbent assay for the presence of Rice tungro bacilliform (RTBV) and Rice tungro spherical (RTSV) viruses. Thus, relationships between the production environment variables and four disease variables — visual incidence and double RTBV and RTSV, single RTSV, and single RTBV infections — were examined. A higher association was observed between site and varieties planted as well as site and synchrony of planting than between site and agroecosystem or site and disease variables (visual incidence, double RTBV and RTSV and single RTSV infections). Disease variables depended on both varieties planted and synchrony of planting and correspondence analysis revealed that the low disease incidence in Nueva Ecija was associated with synchronous planting while the high disease incidence in Isabela was associated with the planting of susceptible varieties and asynchronous planting. Such findings suggest that the relationship between the last two factors at a given site is critical to predicting tungro occurrence. Moreover, correspondence analysis of the relationship among disease variables revealed that tungro incidence is associated with not only double RTBV and RTSV infections but also single RTSV infections. Implications of these results on tungro epidemiology and management are discussed.

Simple Serological Assays for Detecting Rice Tungro Viruses

An attempt was made to produce sensitive and specific polyclonal antisera against the viruses causing rice tungro disease, and to assess their potential for use in simple diagnostic tests. Using a multiple, sequential injection procedure, seven batches of polyclonal antisera against rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) were produced. These were characterized for their sensitivity and specificity using ring-interface precipitin test and double antibody sandwich (DAS) ELISA. Thirty-one weeks after the first immunization, antiserum batch B6b for RTBV showed the highest ring interface titer (DEP = 1:1920). For RTSV, batches S3, S4b and S5b all had similar titres (DEP = 1:640). In DAS-ELISA, however, significant differences among purified antisera (IgG) batches were observed only at IgG dilution of 10-3. At that dilution, IgGB4b showed the greatest sensitivity, while IgGS3 showed greatest sensitivity for RTSV. When all IgG batches were tested against 11 tungro field isolates (dual RTBV-RTSV infections) at sample dilution of 1:10, IgGB4b and IgGB6b for RTBV and IgGS3 and IgGS6b for RTSV performed equally well. However, after cross adsorption with healthy plant extracts in a specially prepared healthy plant-Sepharose affinity column, only IgGB6b could be used specifically to detect RTBV in a simple tissue-print assay.

Agroinoculation Allows the Screening of Rice for Resistance to Rice Tungro Bacilliform Virus

A system for agroinoculating rice tungro bacilliform virus (RTBV), one of the two viruses of the rice tungro disease complex, has been optimised. A nontumour-inducing strain of Agrobacterium (pGV3850) was used in order to conform with biosafety regulations. Fourteen-day-old seedlings survived the mechanical damage of the technique and were still young enough to support virus replication. The level of the bacterial inoculum was important to obtain maximum infection, with a high inoculum level (0.5 × 1012 cells/ml) resulting in up to 100% infection of a susceptible variety that was comparable with infection by insect transmission. Agroinoculation with RTBV was successful for all three rice cultivarss tested; TN1 (tungro susceptible), Balimau Putih (tungro tolerant), and IR26 (RTSV and vector resistant). Agroinoculation enables resistance to RTBV to be distinguished from resistance to the leafhopper vector of the virus, and should prove useful in screening rice germplasm, breeding materials, and transgenic rice lines.

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

Differentiation of Rice Tungro Bacilliform Virus Strains by Restriction Analysis and DNA Hybridization

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

Evaluating Two Mass Screening Methods for Tungro Disease Resistance

Tungro is one of the most destructive viral diseases of rice in South and Southeast Asia. It is associated with two viruses---rice tungro bacilliform virus (RTBV) ,and rice tungro spherical virus (RTSV) (Hibino et al 1978). Both viruses are transmitted by the green leafhopper (GLH) Nephotettix virescens (Ling 1979), However, prior acquisition of RTSV is required for Ihe transmission of RTBV alone (Hibino 1983). Plants infected with both viruses show severe stunting and yellowing. Those infected with RTBV alone show mild stunting but no leaf discoloration whereas those infected with RTSV alone do not show any apparent symptoms (Hibino el al 1978). Since the late 1960s, tungro has been mainly managed through varietal resistance (Khush 1989). The instability of resistant varieties in the field (Dahal et .a1 1990) led to a reexamination of the nature of the incorporated sources of resistance and to the adoption of more precise and more accurate screening methods.

Mechanism of Resistance to Rice Tungro Spherical Virus (RTSV)

RTSV is one of two viruses that cause tungro disease. RTSV is independently transmitted, whereas the other virus, rice tungro bacilliform virus (RTBV), is dependent on RTSV for its transmission by the green leafhopper (GLH), Nephotettix virescens. The occurrence and spread of tungro disease therefore depend on the presence of RTSV in the field. Resistance to RTSV infection would slow down the spread of the disease.

Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease

Background: Multipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo. Design and Methods: We examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major histocompatibility complex-mismatched: UBI-GFP/BL6 [H-2b]→BALB/c [H-2d] and the sibling transplant mimic, UBI-GFP/BL6 [H-2b]→BALB.B [H-2b]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients. Results: Despite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graft-versus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-γ, an important mediator of graft-versus-host disease. Conclusions: Mesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-γ. Our data suggest that both the timing of infusion and the dose of mesenchymal stromal cells likely influence these cells’ effectiveness in attenuating graft-versus-host disease.

Reduced intensity conditioning for allogeneic hematopoietic stem cell transplant determines the kinetics of acute Graft-Versus-Host Disease

Background Preparative myeloablative conditioning regimens for allogeneic hematopoietic stem-cell transplantation (HSCT) may control malignancy and facilitate engraftment but also contribute to transplant related mortality, cytokine release, and acute graft-versus-host disease (GVHD). Reduced intensity conditioning (RIC) regimens have decreased transplant related mortality but the incidence of acute GVHD, while delayed, remains unchanged. There are currently no in vivo allogeneic models of RIC HSCT, limiting studies into the mechanism behind RIC-associated GVHD. Methods We developed two RIC HSCT models that result in delayed onset GVHD (major histocompatibility complex mismatched (UBI-GFP/BL6 [H-2b]→BALB/c [H-2d]) and major histocompatibility complex matched, minor histocompatibility mismatched (UBI-GFP/BL6 [H-2b]→BALB.B [H-2b])) enabling the effect of RIC on chimerism, dendritic cell (DC) chimerism, and GVHD to be investigated. Results In contrast with myeloablative conditioning, we observed that RIC-associated delayed-onset GVHD is characterized by low production of tumor necrosis factor-α, maintenance of host DC, phenotypic DC activation, increased T-regulatory cell numbers, and a delayed emergence of activated donor DC. Furthermore, changes to the peritransplant milieu in the recipient after RIC lead to the altered activation of DC and the induction of T-regulatory responses. Reduced intensity conditioning recipients suffer less early damage to GVHD target organs. However, as donor cells engraft, activated donor DC and rising levels of tumor necrosis factor-α are associated with a later onset of severe GVHD. Conclusions Delineating the mechanisms underlying delayed onset GVHD in RIC HSCT recipients is vital to improve the prediction of disease onset and allow more targeted interventions for acute GVHD.

Developing a therapeutic anti-dendritic cell antibody to prevent graft versus host disease

Host and donor dendritic cells (DC) stimulate alloreactive donor T lymphocytes, and initiate GVHD. We have shown that polyclonal antibody to the DC surface activation marker human CD83 (anti hCD83), which depletes activated DC, can prevent human DC and T cell induced lethal xenogeneic GVHD in SCID mice without impairing T cell mediated anti-leukaemic and anti-viral (CMV and influenza) immunity (J Exp Med 2009; 206: 387). Therefore, we made and tested a polyclonal anti mouse CD83 (RAM83) antibody in murine HSCT models and developed a human mAb against hCD83 as a potential new therapeutic immunosuppressive agent.