Antisera to the nucleoprotein of maize yellow stripe tenui-like virus (MYSV) were produced and used for detection of this virus in several host plants and vector leafhoppers in Egypt. Dot-blot and direct antigen coating (DAC) ELISA were used to detect MYSV in naturally or experimentally infected maize, wheat, barley, oats, and the graminaceous weeds #Bromus wildenowii$, #Cenchrus biflorus$, #Dichanthium annulatum$, #Digitaria sanguinalis$, #Echinochloa colonum$, #Setaria verticillata$ and #S. viridis$. In maize leaves, differences in virus titer appeared to be correlated with leaf age and with MYSV symptom-types. Dot-blot and DAC-ELISA were used also to detect MYSV in naturally or experimentally infective leafhoppers (#Cicadulina chinai$). (Résumé d'auteur

Abol-Ela, S.E.

Ammar, E.D.

Mahmoud, A.

Sewify, G.H.

Thouvenel, Jean-Claude

Horizon / Pleins textes

Phytopath. medit., 1996,35, 19-23 
Detection of maize yellow stripe tenui-like virus by ELISA 
and dot-blot tests in host plants and leafhopper vector in Egypt 
&" P 
P I e. \d A. MAHMOUD, J.-C. HOTJVENEL, S.E. ABoL-ELA *, G.H. SEWIFY * and E.D. AMMAR :$ 
ORSTOM, Giza, Cairo, Egypt 
* Department of Economic Entomology, Cairo University, Giza, Egypt 
Summary. Antisera to  the nucleoprotein of maize yellow stripe tenui-like virus (MYSV) were produced and used 
for detection of this virus in  several host plants and vector leafhoppers in Egypt. Dot-blot and direct antigen 
coating (DAC) ELISA were used to  detect MYSV in naturally or experimentally infected maize, wheat, barley, 
oats, and the graminaceous weeds Bromus wildenowii, Cenchrus biflorus, Dichanthium annulatum, Digitaria 
sanguinalis, Echinochloa colonum, Setaria verticillata and S. viridis. In maize leaves, differences in virus titer 
appeared to be correlated with leaf age and with MYSV symptom-types. Dot-blot and DAC-ELISA were used also 
to detect MYSV in naturally or experimentally infective leafhoppers (Cicadulina chinai). 
Introduction 
The nucleoprotein and non-capsid protein of 
the leafhopper-borne maize yellow stripe virus 
(MYSV), a tentative member of the tenuivirus 
group, have been recently purified and partially 
characterized (Ammar et al., 1990a; Thouvenel 
et al., 1995). MYSV symptom-types include fine 
stripe, coarse stripe, and chlorotic stunt; these 
symptom-types usually appear on different leaves 
of the same plant (Ammar et al., 1990a). MYSV 
was detected, through symptomatology and insect 
transmission, naturally infecting maize, sorghum, 
wheat, barley (Ammar et al., 1989, 1990a), and 
some graminaceous weeds in  Egypt (Sewify, 
1994). It is thought that  wheat, barley and 
graminaceous weeds may play a role in the over 
wintering of both MYSV and its vector leafhopper, 
Cicadulina chinai. However, due to  the lack of 
MYSV antisera, epidemiological studies on this 
disease have so far been very difficult and time 
consuming, depending mainly on the use of 
symptom observation and insect transmission for 
diagnosis. Thus, in the present work, we produced 
specific antiserum to the nucleoprotein of MYSV; 
and we used it, through ELISA and dot-blot 
methods, for detecting MYSV in several host 
plants and weeds as well as in the vector 
leafhopper C. chinai. Additionally, we report 
differences in the virus concentrations that are 
correlated with leaf age and with MYSV 
symptom-types. 
Materials and methods 
S o u r c e s  a n d  m a i n t e n a n c e  o f  
v i r u s  a n d  v e c t o r .  TheMYSVisolates 
and the cultures of the leafhopper vector C. chi- 
nai were the same as those used by Ammar et al. 
(1989, 1990a). MYSV was maintained in maize 
seedlings cv. SC 10 or  DC 204 in a greenhouse 
by serial transmission with C. chinai. Non- 
infective leafhoppers were maintained on caged 
healthy maize plants that were checked regularly 
for virus symptoms. 
P u r i f i c a t i o n  o f  v i r u s  n u c l e o -  
p r o t e i n . The method used for purification 
of the MYSV nucleoprotein was as follows: 
Fonds 
Cote : 
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Documentaire ORSTOM 
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a- 
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infected maize or sorghum tissue (100-200 g of 
leaves or stems) were blended in grinding buffer 
0.2 M Trishoric acid, Polyvinyl Pyrrolidone 2%, 
mercapto-ethanol 0.5% pH 7.0 (1 g/5 ml). After 
grinding for 3-5 min, the extract was filtered 
through cheesecloth and treated with 25% (v/v) 
chloroform. After shaking for 10 min, the 
emulsion was centrifuged at 7,000 rpm for 10 
min. The clarified extract was centrifuged at 
40,000 rpm for 2.5 h at 5°C (Type 55.2 rotor). 
The pellets were resuspended in pellet buffer 
(0.01M KH,PO,, 0.01M Ethylene diamine 
tetraacetic acid (EDTA), pH 7.0) and kept 
overnight at 4°C or  directly dissolved, stirred and 
then layered onto 6 ml of 20% sucrose (w/v) in 
pellet buffer and centrifuged at 40,000 rpm for 3 
h at 5°C. Pellets were resuspended in  the pellet 
buffer and kept overnight at 4°C. The suspension 
was clarified and layered on 10-40% sucrose 
density gradients in phosphate buffer (0.01M 
KH,PO,, pH 7.0) and centrifuged at 25,000 rpm 
for 4 h at  5°C (Beckman SW 28 rotor). One zone 
was observed by scanning at  260 nm wavelength, 
diluted in distilled water and pelleted at 40,000 
rpm for 2 h at 5°C then resuspended in phosphate 
buffer. 
P r o d u c t i o n  a n d  t i t r a t i o n  o f  
a n t i s e r u m . Two New Zealand white 
rabbits were immunised by three weekly 
intramuscular injections of purified MYSV 
nucleoprotein emulsified with an  equal volume 
of Freund's complete adjuvant for the first and 
second injections, and with incomplete adjuvant 
for the third injection. Blood was collected on the 
10th day after the third injection. 
The titre of the antiserum produced to  the 
virus nucleoprotein was estimated by agar gel 
double diffusion tests (titre 1/64). Infected maize 
tissue was used as antigen. It was ground in 
0.9% NaCl (lg/lO ml), and squeezed through two 
layers of cheesecloth. The antiserum was first 
cross-absorbed by mixing with healthy maize 
leaves extract, and then centrifuged at 7,000 rpm 
for 10 min. The pellet was discarded and the 
supernatant used as antibodies fraction. 
E n z y m e - l i n k e d  i m m u n o s o r b e n t  
a s s a y ( E L I S A 1 t e s t . A direct antigen 
coating (DAC) ELISA was used as described by 
Converse and Martin (1990) with a few 
modifications. Test samples were ground with a 
homogenizer in coating buffer pH 9.6 (1 g/10 ml) 
applied to  the microtiter plate and incubated 
overnight at 4°C. Wells were washed with PBS- 
Tween buffer 3 times, then a blocking agent (5% 
non fat dry milk powder) was applied at  room 
temperature for 30 min. Antiserum was then 
added at the dilution of 1/1,000 in PBS-Tween 
buffer pH 7.4 containing 2% polyvinyl- 
pyrrolidone-40 (PVP-40) and incubated for 3 h at 
37"C, or overnight at 4°C. The plate was washed, 
dried and the conjugate to  anti-rabbit alkaline 
phosphatase, diluted at 1/2,000 in conjugate 
buffer containing ovalbumin 0.2 g/l in PW-PBS- 
Tween pH 7.4 was added and incubated 3 h at 
37°C or overnight at 4°C. The plate was then 
washed and dried. The p-nitrophenyl phosphate 
(PNPP) substrate was dissolved in substrate 
buffer pH 9.8 (1 mg/ml). Absorbance values were 
measured at  405 nm wavelength. Absorbance 
values more than twice those of healthy controls 
were considered positive. 
D o t - b l o t  i m m u n o a s s a y s  o f  
p l a n t s  a n d  l e a f h o p p e r s .  The 
method used was that of Lin et al. (1990) with a 
few modifications. For some experiments nylon 
membrane (0.45 ym pore-Amersham), or 
preferably nitro-cellulose membrane (0.20 pm-Bio 
Bind-NC-Whatman) was directly used. Tissue 
extract (0.1 g infected maize plant/l ml buffer or 
1 insect/lO y1 buffer) were applied on the mem- 
brane after grinding in PBS buffer, pH 7.3 
followed by clarification. The membrane was dried 
for at least 5 min, immersed in PBS-Tween 
containing 5% non fat dry milk, incubated for 45 
min at 37"C, shaken for 15 min. It was then 
washed with PBS-Tween, for 3 times by shaking 
it (2-5 min each), incubated with antiserum (cross- 
absorbed) at a dilution of 1/1,000 in PBS-Tween 
for 2 h at 37"C, or overnight at 4"C, shaken for 
15 min, then washed as above. The membrane 
was later incubated with conjugated anti-rabbit 
alkaline phosphatase diluted at 1/2,000 in PBS- 
Tween for 2 h a t  37"C, washed as above and 
then incubated with substrate containing 30 pl 
BCIP, 30 pl NBT/15 ml substrate buffer, pH 9.8. 
The membrane was washed by distilled water to  
stop the reaction. All incubations were done in 
the dark. 
H o s t  p l a n t  s t u d i e s .  Theabove 
mentioned serological methods were used to 
detect MYSV in  naturally or experimentally 
infected winter host plants and graminaceous 
weeds. During October 1990, june 1991 and apri1 
1993, diseased weeds showing symptoms similar 
20 
t o  those of MYSV were collected from the farm of 
the Faculty of Agriculture, Cairo University, at 
Giza, Egypt. These weeds were: Digitaria 
sanguinalis and Setaria virìdis. 
Additionally, the following plants were 
experimentally inoculated, by feeding on them 
MYSV-infective leafhoppers for 1-7 days (5 
insects/plant): maize (2. mays L.), wheat (T. 
sativum L.), barley (Hordeum vulgare L.), oats 
(Avena fatua L.), and the graminaceous weeds 
B r o m u s  wildenowii, Cenchrus bif lorus,  
Dichanthium annulatum, Echinochloa colonum 
and Setaria verticillata. These plants were 
checked serologically for virus presence 8 weeks 
after the inoculation. 
The virus titer was assessed by DAC-ELISA 
in different parts of infected maize plants (leaves, 
stems and roots) as well as in leaves of different 
,ages showing different symptom-types. 
A t t e m p t s  t o  s e p a r a t e  M Y S V  
s y m p t o m - t y p e s . Three types of MYSV 
symptoms that occur on naturally infected plants, 
i.e. fine stripe, coarse stripe and chlorotic stunt, 
were previously recorded (Ammar et al., 1989, 
1990). In a trial to separate these symptom types, 
healthy C. chinai leafhoppers were starved for 1- 
2 h before virus acquisition feeding for 15, 20, 30 
or 35 min on pieces of maize leaves showing only 
one type of symptoms. Insects were then  
transferred to  healthy maize seedlings for 1 week 
to allow for virus incubation in the vector. 
Leafhoppers were then placed on maize seedlings 
(10 insects per plant) for 1-2 wks inoculation tests. 
The symptom-type was recorded on each test 
plant 2-3 weeks later. 
Results 
S e r o l o g i c a l  t e s t s  o n  p l a n t s .  
MYSV was detected by either DAC-ELISA or dot- 
blot in naturally infected maize and graminaceous 
weeds Digitaria sanguinalis and Setaria viridis. 
It was also detected serologically i n  
experimentally inoculated maize, wheat, barley, 
oats, and  in  the  graminaceous weeds B. 
wildenowii, C. biflorus, D. annulatum, E. colonuna 
and S. verticillata. 
In DAC-ELISA, MYSV antiserum had a titer 
of with leaf extracts or purified virus (O.D. 
1.39) diluted to (Fig. 1). In dot blot tests, the 
titer for MYSV antiserum was 
3's T 
1 o-' 1 0.5 
MLUTIONS 
Fig. 1 - Detection of MYSV in maize plants using Direct 
Antigen Coating - ELISA method. Antigen was diluted (extract 
and purified virus - 0,4 O.D. -1 in coating buffer. Absorbance 
values were recorded after 30 min. 
D e t e c t i o n  i n  t h e  l e a f h o p p e r  
v e c t o r . Using DAC-ELISA and the MYSV 
antiserum, the virus was detected in clarified extracts 
of infktive C. chinai leafhoppers at a dilution of 103 
(Fig. 2). Using the dot-blot method, MYSV was 
detected in c l d e d  extracts of single infective insects 
at the dilution of U4 (single insects). 
C o r r e l a t i o n  o f  M Y S V  t i t e r  
w i t h  l e a f  a g e  a n d  s y m p t o m - t y p e .  
The three types of MYSV symptoms, i.e. fine 
stripe, coarse stripe and chlorotic stunt usually 
appear at this order on different leaves of MYSV- 
infected maize plants. However, the chlorotic 
stunt type usually appears only on naturally- 
infected field plants. Attempts to  separate these 
Fig. 2 - Detection of MYSV in the leafhopper vector using 
Direct Antigen Coating - ELISA method. Antigen was diluted 
(extract) in coating buffer. Absorbance values were recorded 
after 30 min. 
21 
Y 
symptom-types through short acquisition access 
period (AAP) of 15, 20 and 30 min of the 
leafhopper vector C. ckinai on leaf pieces showing 
only one symptom-type were unsuccessful. No 
transmission occurred following the 15 min AAP, 
whereas both fine and coarse stripe resulted from 
longer AAP on leaves showing fine stripe, coarse 
stripe or chlorotic stunt. 
Using DAC-ELISA with MYSV nucleoprotein 
antiserum, MYSV titer was assessed in different 
parts of infected maize plants including stems, 
roots, and leaves of different ages showing 
different symptom types. As indicated in Fig. 3, 
MYSV titer was higher in both leaves and stems 
then in the roots of infected maize plants. Also, 
virus titer was higher in younger leaves showing 
coarse stripe, than in older ones showing fine 
stripe. 
Disc us s i o n 
Previous studies on MYSV had to rely mainly 
on symptomatology and the laborious and time 
consuming insect-transmission tes ts  for 
identification of this virus in different host plants 
in Egypt (Abou1 Ata and Ammar, 1985; Ammar 
et al., 1984, 1987, 1989; Sewify, 1994). Production 
and use of specific antiserum to  the nucleoprotein 
of MYSV in the present work will facilitate 
further epidemiological studies on this virus and 
its vector in Egypt. 
Furthermore, it will help to  identify this virus 
or other viruses serologically related to it in other 
maize-producing countries particularly in Africa 
or in the Middle East. In  our study, both DAC- 
0.8 
0.7 
E 0.6 
a 
* 0.5 
5 
0.4 
d 
$, 0.3 
P; 
0.2 
0.1 
O 
Fig. 3 - Detection of MYSV in different parts of the same 
plant by Direct Antigen Coating-ELISA method. Absorbance 
values were recorded after 30 min. 
ELISA and dot-blot methods were successful in 
detecting low concentrations of MYSV in host 
plants or  in leafhopper vectors. DAC-ELISA is 
more economic and less complicated than other 
ELISA methods and is thus more useful in field 
surveys. However, the dot-blot method may be 
more sensitive for detecting MYSV in single 
leafhoppers. Either method can be used for futu- 
re studies to  determine if MYSV replicates in its 
leafhopper vector, which is a crucial characteristic 
for the classification of MYSV as a tenuivirus 
(Gingery, 1988; Ammar et al., 1990a). 
In the present study, using either DAC-ELISA 
or dot-blot serological methods, we confirmed and 
added to the previously recorded host range of 
MYSV. In Egypt, MYSV has been found naturally 
infecting maize, sorghum and wheat plants 
(Ammar e t  al., 1990) as  well as some 
graminaceous weeds (Sewify, 1994). Furthermore, 
MYSV can experimentally infect barley (Ammar 
et al., 1989), oats and several other graminaceous 
weeds. It has been suggested that wheat, barley, 
and some graminaceous weeds may favour the 
overwintering of MYSV and its leafhopper vector 
in Egypt (Ammar et al., 1989; Sewify, 1994), but 
further work is still required. 
The three symptom-types associated with 
MYSV, i.e. fine stripe, coarse stripe and chlorotic 
stunt, usually appear in this order on successive 
leaves of the same maize plant (Ammar et al., 
1989; 1990a). These symptom-types could not be 
separated using short acquisition feeding periods 
of the vector (Ammar et al., 1989; and present 
study). Electron microscopy of thin sections of 
maize leaves revealed no  differences in the 
cytopathology and ultra structure o f  leaves 
showing any of these symptom-types (Ammar et 
al., 1990a). Here in DAC-ELISA tests, MYSV titer 
was higher in younger maize leaves showing 
coarse stripe, than in older leaves showing fine 
stripe. These results suggests t ha t  MYSV 
symptom-types may be correlated with both leaf 
age and virus concentration in maize leaves. It is 
possible that the first leaves to  be infected may 
get lower concentration of MYSV and thus 
develop fine stripe, whereas younger leaves may 
get higher concentration of virus and thus develop 
coarse stripe or chlorotic stunt. However, more 
serological studies, that take in consideration the 
plant and leaf ages during infection under field 
as well as greenhouse conditions, are needed to  
substantiate and expand this hypothesis further. 
22 
1 
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Detection of maize yellow stripe tenui-like virus by ELISA and dot- blot tests in host plants and leafhopper vector in Egypt

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