Ann. appl. B i d . (1994), 125, 13S140 Printed in Great Brilain 133

Serological relationships among rice yellow mottle virus isolates

By A N MANSOUR and K W BAILLIS* Department of Plant Protection, Faculty of Agriculture, University of Jordan,

Amman, Jordan * Brialynn, Bowes Road, Mersham, Ashford, Kent TN25 6NN, U K

(Accepted 4 March 1994)

Summary Serological studies on five isolates of RYMV collected from the Ivory Coast

(IC), Sierra-Leone (SL), Niger (Nr), Kenya (K) and Nigeria (N) indicated that these isolates are serologically related. Gel double diffusion and direct ELISA tests showed that the five isolates could be arranged into three serological groups here designated RYMV-N, SL-IC and K-NR. However, the ISEM studies did not reveal any clear grouping of the heterologous isolates tested.

Key words: Rice yellow mottle virus, Africa, serological relationships

Introduction Rice yellow mottle virus (RYMV), which occurs only in Africa, is a major problem in

irrigated rice areas in West and East Africa. Although much effort has been made at the International Institute of Tropical Agriculture (IITA) in Nigeria to develop high-yielding rice cultivars with resistance to RYMV (Rossel, 1986), little attention has been given in breeding programmes to the relationships between isolates of RYMV from different regions. Knowledge of the serological relationships between RYMV isolates is valuable in diagnostic work and may prove to be important in epidemiological studies and disease control. In this study serological comparisons were made for five isolates of RYMV.

Materials and Methods

Virus source RYMV isolates from Africa were kindly supplied by C Fauquet from the Ivory Coast

(RYMV-IC), L Bos from Kenya (RYMV-K), D Lesemann from Niger (RYMV-Nr), G Thottappilly from Nigeria (RYMV-N) and S N Fomba from Sierra-Leone (RYMV-SL).

The isolates were recovered from rice leaves by mechanical inoculation and maintained in rice cv. ITA 212. Inoculum was prepared in 0.067 M neutral Sorensen's buffer (1:lO w:v) containing 1 m d m l Celite. Inoculation was made by rubbing both surfaces of leaves at the 4 4 leaf stage. Test plants were kept under glasshouse conditions (25-26°C) at Wye College, Wye, Ashford, Kent, UK. When necessary in winter months, supplementary illumination from high pressure sodium lamps was used. 0 1994 Association of Applied Biologists

134 A N MANSOUR AND K W BAlLLlS

Purification RYMV was purified according to the method of Hull (1977), Denlyo, Homer & Hull

(1978) and Brisco, Hull & Wilson (1985) for the purification of subemoviruses. Systemically infected leaves were harvested 1 wk after symptoms first appeared, but

before leaves turned necrotic, and stored at 20°C. Frozen leaves ( 5 gm) were homogenised in a Sorvall Omni-mixer for 2 min with 50 ml of 0.1 M sodium acetate buffer (pH 4.8). The homogenate was strained through two layers of muslin and the pulp was re-extracted in a further 50 ml buffer. The sap was subjected to centrifugation at 10 000 g for 15 min at 4°C (RC5C Sorvall centrifuge, Dupont). Polyethylene glycol (PEG 6000) and NaCl were slowly added to the supernatant while stirring to give final concentrations of 10% and 1% (w/v), respectively. After stirring for 1 h at 4"C, the suspension was centrifuged at 10 000 g for 15 min. The pellet was resuspended in 10 ml of 0.1 M sodium acetate (pH 5.0). Following low speed centrifugation, the supernatant was centrifuged at 101 000 g for 2 h in a TFT 70.38 rotor at 4°C using a Centrikon Ultracentrifuge. The pellet was resuspended overnight in 2 mlO.1 M sodium acetate (pH 5.0) and clarified by low speed centrifugation. Infectivity was tested by inoculating rice cv. ITA 212 plants. Absorbance was determined using a SP8-100 spectrophotometer. Virus concentration was estimated assuming the extinction coefficient of RYMV to be 6.5 (Baker, 1974). The partially purified virus was stored at 4°C with 0.02% (w/v) chlorobutanol (Hull, 1985).

Agar gel double diffusion test Tests were performed in plastic Petri dishes with 1% Ion agar No. 2 in distilled water

containing 0.85% NaCl and 0.2% sodium azide. Antisera to RYMV-N, K and IC were kindly provided by Drs G Thottappilly (IITA, Nigeria), D Peters (Agricultural University, Wageningen) and C Fauquet (ORSTOM, Ivory Coast), respectively. Antisera were diluted 1:32 (v/v) with 0.85% NaCl. Antigens were prepared by grinding infected leaves (1:l w:v) in 0.85% NaCl.

Imrnunosorbent electron microscopy ( ISEM) The procedure used as based upon the methods of Derrick (1973) and Milne (1986).

Fresh formvar coated grids were each floated on a 15 pl droplet of the globulin fraction (1 mg/ml) diluted 1:lOO (v/v) with Sorensen's buffer (pH 7) for 5 min at room temperature (Milne, 1986). After washing with Sorensen's buffer and draining, individual grids were placed on 20 p1 droplets of test leaf extract for 15 min. Grids were washed and drained and then floated on a 15 pl droplet of gamma globulin for 5 min at room temperature.

The decorated grids were washed with buffer and distilled water and then stained with 2% uranyl acetate and examined in a Hitachi H 7000 electron microscope. Leaf extracts were prepared by macerating infected tissue (1: 100 w/v) in 0.1 M Sorensen's buffer (pH 7). The extract was squeezed through muslin and subjected to low speed centrifugation (4200 g for 5 min) before use.

ELISA The direct double antibody sandwich, enzyme linked immunosorbent assay (DAS-

ELISA) method was essentially used as described by Clark & Adams (1977) and Hill (1984). For each experiment at least two replicate wells per treatment were used, and each experiment was repeated twice. Absorbance values presented in the Results are the average of two experiments. The tests were made with serial half-log,o dilutions of partially purified preparations of all five RYMV isolates and with serial 10-fold dilutions of sap extracts from ITA 212 RYMV infected leaves. Antisera to RYMV-N. K and IC were used. Extracts of

Serological relationships among RYMV isolates 135

healthy leaves were always included. Gamma globulins were used at 1 mg/ml and alkaline phosphatase conjugates were used at a dilution of 1:1500. Absorbance at 410 nm was measured 30 min after the addition of substrate.

Results Purification

The purification procedure was apparently satisfactory in terms of rapidity, ease, as well as in virus production and removal of host contaminants.

The concentration of the virus in leaves of the susceptible rice cv. ITA 212 was very high. A yield of 0.5 mg/g, 0.75 mg/g and 1 mg/g was recovered with RYMV-SL and IC, N and Nr, and K isolates, respectively.

Agar gel double-diffusion tests Tests were made with antisera prepared against N (Fig. lA), K (Fig. 1B) and IC (Fig.

1C) isolates. With each antiserum, four plates were used. Antigens were arranged so that each was placed between two of the other four antigens. This allowed 10 combinations of antigen pairs to be tested against each antiserum (Fig. 1; Table 1). With all three antisera, when antigens K and Nr were in adjacent wells spur formation did not occur and precipitin lines fused (Fig. 1). When antigens IC and SL were placed in adjacent wells, precipitin lines fused when tested with antisera to K and IC (Fig. lB , 1C) but, with RYMV-N antiserum, antigen SL spurred over antigen IC (Fig. 1Ad).

When IC, SL, Nr and K antigens were tested against RYMV-N antiserum, spurs always occurred between wells containing antigen SL and the other three isolates with SL spurring over antigens K, Nr or IC (Fig, 1A).

When antigens IC, SL and N were tested with RYMV-K antiserum (Fig. lB), antigens IC and SL spurred over antigen N, indicating that there were K antibodies common to both isolates IC and SL and another reacted with antigens IC or SL to produce the spurs.

ISEM Results of ISEM tests showed that particles of all RYMV isolates, whether treated with

homologous or heterologous antibody, became decorated to some extent (Fig. 2). It was difficult to discern consistent and clear differences in the intensity and extent of the antibody halo around virus particles. However, there was more extensive decoration around the homologous compared with heterologous virus for both N and IC antiserum (Fig. 2A, 2B).

ELISA ELISA test results showed that all five isolates of RYMV were detected in both purified

preparations and crude sap extracts when tested against antisera to RYMV-N, -K and -1C (Fig. 3A, 3B).

When extracts were used, RYMV-N antibodies reacted most strongly with the hom- ologous antigen giving high absorbance values; absorbance was off scale for sap diluted 1:lO to 1:lOOO (Fig. 3Aa). RYMV-N antibodies reacted least with the heterologous Nr and K antigens; absorbance values were very similar for these two antigens. RYMV-N antibodies produced similar intermediate absorbance values with antigens IC and SL. Clearest dis- crimination between absorbance and antigen was seen at saturating antigen concentration.

136 A N MANSOUR AND K W BAILLIS

Fig. 1. Central well: (A) N RYMV-N antiserum, (B) K RYMV-K antiserum, ( C ) IC RYMV-IC antiserum. Peripheral wells: Extracts from rice (cv. ITA 212) infected with isolates N (Nigeria), K (Kenya). SI. (Sierr-Leone). NR (Niger) and IC (Ivory Coast).

Reaction with RYMV-IC antiserum separated the isolates into two groups (Fig. 3Ab). High absorbance values were obtained with the homologous RYMV-IC and the hetero- logous RYMV-SL antigens, whereas isolates N , Nr and K reacted less strongly but were very similar to each other (Fig. 3Ab). Two groupings were also evident with RYMV-K antiserum (Fig. 3Ac), with which isolates RYMV-K and Nr induced high absorbance values,

Serological relationships among R Y M V isolates 137

Fig. 2 . A. a) Homologous isolates RYMV-N, b) Heterologous RYMV-SL, c) Heterologous RYMV- Ic , d) Heterologous RYMV-K, e) Heterologous RYMV-Nr. B. a) Homologous isolates RYMV-IC, b) Heterologous RYMV-SL, c) Heterologous RYMV-N, d) Heterologous RYMV-K, e) Heterologous RYMV-Nr .

whereas values for antigens N, IC and SL gave values lower than the K and Nr group. In all tests, the differences between the isolates were clear up to a sap dilution of c. 1 : l O 000.

Trends were essentially similar when serial half-loglo dilution of partially purified antigen preparations were used (Fig. 3B), except the antigen saturation was only apparent for

Table 1. A summary of the reactions illustrated in Fig. 1 between antigen pairs for all combinations of f ive R Y M V isolates tested against antisera to RYMV-N, -IC and -K in agar

double-d$fusion tests

Adjacent antigen pairs

N-IC N-SL N-K

IC-SL IC-K IC-Nr Nr-K Nr-SL K-SL

Antisera to r , RYMV-N RYMV-IC RYMV-K

Na N N SL

- SL SL

IC SL

b - - IC IC

SL SL

IC SL K

K Nr

-

Nr K

aLetters indicate which antigen of each adjacent pair precipitation line extention to form a spur. bPrecipitation lines fused.

138 A N MANSOUR AND K W BAILLIS

antiserum O/SJ .-*-*

.F

A

r 0 - 0 - 0 b RYMV-IC DI--P(--R c RYMV-K r-

" 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 I 0 1 2 3 4 5 6 I

Reciprocal of sap dilution (log,,)

B

b RYMV-IC c RYMV-K ' f- '-1 antiserum

500 158 50 15.8 5.0 1.58 500 158 50 15.8 5.0 1.58 500 158 50 15.8 5.0 1.58

Virus concentration (ngiml)

Fig. 3. Comparison of the five isolates of RYMV by double antibody sandwich ELISA: a) RYMV-N. b) RYMV-IC, c) RYMV-K. A. Crude sap extracts. B. Purified virus antigens.

antigens K and Nr tested against RYMV-K antigen (Fig. 3Bc). In all instances, homologous combinations induced the highest absorbance values together with heterologous antigens SL and Nr when tested against RYMV-IC and RYMV-K antisera, respectively. Isolates could be grouped as in the crude sap tests but, with partially purified antigen preparations. antigen RYMV-N was more clearly separable from antigens of the other four isolates.

Discussion The presence of a strong precipitin line between the three antisera tested and the five

isolates of RYMV indicated that all were serologically related. However, spur formation between antigen N and the other four RYMV isolates when RYMV-N antiserum was used

Serological relationships among R YMV isolates 139

suggested isolate N was antigenically distinct from others. Using RYMV-N antiserum, antigen SL always formed a spur when placed in a peripheral well adjacent to antigen IC, Nr and K (Table 1). This suggested that isolate SL has more determinants in common with isolate N than the remaining three isolates. When isolates were tested against RYMV-IC antiserum, a spur formed between antigen IC and N, Nr, or K antigens suggesting that isolate IC was serologically distinct from isolates N, Nr and K. Fauquet & Thouvenel(l977) reported spur formation when isolates from the Ivory Coast and Bakker’s (1974) original Kenyan isolate were tested using either RYMV-IC or -K antiserum. Although IC and SL antigens formed a fused line when RYMV-IC and RYMV-K antisera were used, the presence of spur between them when RYMV-N antiserum was used indicated slight differences between the two antigens.

The absence of spur production between antigens K and N, irrespective of the antiserum used, was strong evidence that the two isolates were serologically indistinguishable (Table

When the antigen pairs N and IC, and N and SL were placed in adjacent wells and tested against RYMV-K antiserum (Table l), antigens IC or SL formed spurs, suggesting that they have more determinants in common with isolate K than isolate N. However, it was not clear which isolate was more closely related to isolate K as RYMV-SL antiserum was not available. Based on the present gel diffusion results, the five isolates of RYMV can be arranged into three serological groups, designated here RYMV-N, SL-IC and K-Nr.

When crude leaf saps were tested against RYMV-IC and RYMV-K antisera, the isolates could be divided into two groups (Fig. 3Ab; 3Ac). However, when RYMV-N antisera were used, isolates could be categorised into three different groups as suggested by the gel double diffusion results. The same three groups were evident when purified preparations were used in ELISA tests using any of the three antisera available (Fig. 3B). However, in double diffusion tests, it was not possible to categorise the isolates of RYMV using one antiserum, whereas in ELISA tests with purified preparations it was possible to group the isolates into three different groups. This could be explained by the fact that ELISA is more sensitive than double diffusion tests (Thomas, 1980).

There was no obvious relationship between serological properties and the geographical origin of RYMV isolates. For example, it found that the Kenyan (East Africa) and the Niger (West Africa) isolates were serologically indistinguishable, whereas the Nigerian and Niger isolates were serologically distinct, although the two countries have an extensive common boundary.

1).

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(Received S May 1993)