Tungro screen kits for extension agents and plant breeders

O. Azzam, L. Kenyon, and P.D. Nath

Abstract

For the last 14 years, IRRI has used the enzyme-linked immunosorbent assay (ELISA) method to screen and evaluate rice germplasm for tungro resistance and tolerance. Unfortunately, due to a lack in resources and technical capacities, this technology has not been taken up by national programs. Two years ago, collaborative activities between IRRI and NRI were initiated to develop diagnostic kits that are simpler and more suitable to the needs of national breeding programs and extension services. In this study, we report on the successful development of a Tungro Screen Kit for rice tungro bacilliform virus, one of the two viruses that cause the tungro disease. Five prototype Tungro Screen B kits were assembled and distributed at the Tungro Management Workshop held at IRRI in November 1998 for field testing in India, Indonesia, and the Philippines.

Introduction

In Southeast Asia, the diagnosis of the two viruses associated with tungro disease, rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV), relies mainly on the specific orange-yellow leaf discoloration symptoms exhibited on susceptible cultivars. Several of these cultivars, however, do not produce the specific symptoms in the field or under experimental conditions. Furthermore, if plants are infected with RTSV only, there are no, or only very mild, symptoms.

When enzyme-linked immunosorbent assay was introduced at IRRI in 1985, it revolutionized the approaches to studying the ecology and epidemiology of these two viruses by providing a relatively fast and accurate means of detection. For the last 14 years, IRRI has used the technique to screen and evaluate rice germplasm for tungro resistance and tolerance. More than 35,000 rice accessions have been evaluated and potential sources of resistance against RTSV and tolerance for RTBV have been identified.

In addition to screening germplasm, IRRI assists national research programs in their studies on tungro disease. Unfortunately, because of a lack of resources for establishing and maintaining such facilities and technical difficulties in producing the antisera locally, and optimizing and troubleshooting the technique routinely, the ELISA procedure has not been taken up by the national programs. Commercially produced kits based on 96-well ELISA plates and specific antisera against RTBV and RTSV are available (e.g., Adgen), but these are generally too expensive for routine use in breeding programs or by resource-poor extension services. They also usually require some expertise and the use of expensive equipment for reliable assessment of results.

This report summarizes the collaborative activities undertaken by IRRI and the Natural Resources Institute to try to produce antisera with high titer and specificity suitable for use in simple diagnostic kits by resource-poor national programs and extension services.

Materials and methods

Batches of antisera against the two viruses were produced by using a modification of the usual procedure. Instead of giving the rabbits only three intermuscular and one interveinal injection of

the purified virus particles (in adjuvant) at weekly and biweekly intervals, they were given nine, with 6-wk intervals between each of the later seven injections. Approximately 20 mL of blood were collected from each rabbit 10 days after each of the later seven antigen injections. This immunization regime is reported to increase the titer and specificity of the resulting antisera (Harlow and Lane 1988).

Antisera titers were first measured using purified virus particles in the ring-interface precipitin test (Van Regenmortel 1982). Immunoglobulins were purified from the sera by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography. Alkaline phosphatase was conjugated to the immunoglobulins by the glutaraldehyde procedure (Clark and Adams 1977). The titer and specificity of each of the different batches of purified immunoglobulins were tested in double antibody sandwich (DAS)-ELISA using IgG to trap and IgG-alkaline phosphatase to detect the trapped virions.

The batches of antiserum with the greatest titer and specificity were tested in a non-quantitative membrane-based “tissue-print’’ assay for their suitability for use in a simple diagnostic kit. Freshly cut stems and leaf midribs were “printed” onto the surface of nitrocellulose or polyvinylidene fluoride (PVDF) membranes. The membranes were then probed either with the virus-specific antisera batch followed by a commercial alkaline phosphatase-labeled anti-rabbit immunoglobulin or directly with the virus-specific antibody batch conjugated to alkaline phosphatase. Signal development in either case was by using the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate.

Results and discussion

The need by national programs and extension agents for simple, cheap, and reliable diagnostic tools for rice tungro viruses had been expressed frequently (e.g., Foot 1995). In 1997, Cabauatan and Koganezawa described a simple assay based on the use of colored latex beads bound to specific polyclonal antisera. This procedure, however, was not taken up to any great extent, probably because of the requirement to homogenize and centrifuge samples, and the large quantities of antiserum the test required.

Of the batches of antisera produced against RTBV and RTSV by the repeated immunization method, RTBV-6 and RTSV-6 showed the greatest titers in the ring interface test. All seven batches for each virus had sufficient specificity and titer for use in DAS-ELISA, although they did present relatively high healthy-background absorbances. This high level of cross-reaction to components from healthy rice plants meant that none of the antisera could be used directly in tissue-print assays because the difference in signal strength between infected and healthy plants was often too small to be reliably observed by the eye.

To try to overcome the problem of cross-reaction to healthy plant components, the batches of antiserum with the greatest titer and least cross-reactivity were cross-adsorbed with healthy sap components. The most effective method for doing this was by first passing the immunoglobulin fractions through a specially prepared healthy rice- proteins-sepharose column (prepared by treating cyanogen-bromide-activated sepharose with homogenate from healthy rice plants). The resulting solution was then incubated with a piece of nitrocellulose membrane previously saturated with healthy rice plant homogenate. This mopped up any remaining antibodies with affinity for plant proteins.

After this double-cross adsorption, only RTBV-IgG batch 6 retained sufficient titer to be used effectively with little healthy-background reaction in tissue-print assays (Fig. 1). This doubly

cross-adsorbed antibody is the basis for the prototype Tungro Screen B diagnostic kits. Because a helper component from RTSV is required for vector transmission of RTBV, a positive reaction with the RTBV detection kit in the field implies the presence of RTSV as well. A simple diagnostic test for the presence of RTSV alone, however, would still be useful.

Five prototype Tungro Screen B kits were distributed at the November 1998 Tungro Management Workshop held at IRRI for field testing in India, Indonesia, and the Philippines. Included with each kit was a question sheet to be filled out and returned to IRRI with the test membranes. The questionnaire asked for details about rice varieties tested, plant age, how plants were stored prior to testing, if there was a delay between printing and developing the membranes, and how easy the evaluator found the test procedure, and comments about the kit or its possible improvement. Based on these responses, it is anticipated that modifications or improvements to the kit will be made in the near future.

References

Clark MF, Adams AN. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34:475–483.

Cabauatan P, Koganezawa H. 1997. Alternative methods for detection of rice tungro viruses. In: Chancellor TCB, Thresh JM (editors). Epidemiology and management of rice tungro disease. Chatham (UK): Natural Resources Institute.

Foot C. 1995. A diagnostic kit for the rapid detection of tungro in the field. NRI-IRRI internal report.

Harlow E, Lane D. 1988. Antibodies, a laboratory manual. New York (USA): Cold Spring Harbor Laboratory Press.

Van Regenmortel MHV. 1982. Serology and immunochemistry of plant viruses. New York (USA): Academic Press. 302 p.

NotesAuthors’ addresses: O. Azzam and P.D. Nath, International Rice Research Institute, MCPO Box 3127, Makati City 1271, Philippines; L. Kenyon. Natural Resources Institute, University of Greenwich, Central Avenue, Chatham, Maritime, Kent ME4 4TB, UK.

Citation: Azzam O, L. Kenyon, and P.D. Nath. 1999. Tungro screen kits for extension agents and plant breeders. p. 76-80. In: Chancellor TCB, Azzam O, Heong KL (editors). Rice tungro disease management. Proceedings of the International Workshop on Tungro Disease Management, 9–11 November 1998, International Rice Research Institute, Los Baños, Philippines, 166 p.