Mahuku, George S.

Toler, R.W.

Texas A&M University

Z TA24S.7 
8873 :--------------,1 
NO.1706 8-1706 May 1992 
TEXAS A&M UNIVERSITY LIBRARY 
Effect of Inoculation Pressure 
. on Maize Dwarf Mosaic Virus Strain-A 
Disease Incidence, Severity 
and Titer in Sorghum 
The Texas Agricultural Experiment Station • J. Charles Lee, Interim Director 
The Texas A&M University System • College Station, Texas 
~ • . k ( :_ 
., 
., 
(Blank Page in OrigiBatBuUetiol 
I 
.I 
,. : ::; , :" 
Effect of Inoculation Pressure on Maize Dwarf 
Mosaic Virus Strain-A Disease Incidence, Severity 
and Titer in Sorghum 
George s. Mahuku and R. W. Toler* 
* Plant Protection Research Institute, Department of Research and Specialist Services, P.O. Box 8100, 
Causeway, Harare, Zimbabwe, and Professor, Department of Plant Pathology and Microbiology, Texas 
Agricultural Experiment Station, Texas A&M University, College Station, TX 77843 
." 
Abstract 
Use of a spray gun for mechanical inoculation of 
viruses could result in different pressures during 
inoculation, affecting disease ratings of two sorghum 
cultivars. This experiment evaluated the effect of 
mechanical inoculation pressure on virus titer as 
measured by double antibody sandwich method of 
enzyme-linked immunosorbent assay (DAS-ELISA), 
disease incidence and severity in sorghum cultivars 
differing in reaction to maize dwarf mosaic virus 
strain-A (MDMV-A). Positive correlations (r=0.58 
and r=.0.53 for RTx430 and BTx378 respectively), 
were obtained between disease incidence (percentage 
infection) when inoculation pressure was in the range 
of 5.28 to 7.06 kg/cm2. Neither disease severity nor 
titer was correlated to inoculation pressure. How-
ever, inoculations resulted in higher virus titer com-
pared to uninoculated plants. 
Introduction 
Maize dwarf mosaic virus (MDMV) occurs world 
wide in gramineous hosts and causes significant 
economic loss in several crops, including grain and 
forage sorghum, millet, and maize. Host plant resis-
tance has reduced the losses caused by this virus 
disease. 
Artificial inoculation (using an airgun) of sorghum 
with MDMV has led to the release of tolerant and 
resistant inbreds and hybrids. When a spray gun is 
used for mechanical inoculation, differences in pres-
sures during inoculation may result in selectively 
affected disease ratings among cultivars. Different 
field inoculation techniques have been used (6, 7,8, 
9), and there is no consistency in the protocol fol-
lowed. Inoculation pressures ranging from 4 kg/cm2 
to 8.5 kg/cm2 (6, 8) have been used for field inocula-
tions. There has been no mention of the effect of 
inoculation pressure on virus titer and yield. Toler 
and Miller (15), using four different inoculation pres-
sures (0, 1.4, 4.2, and 7.3 kg. cm2), found increased 
cultivar susceptibility with increased pressure, and 
disease severity varied under different pressures. In 
their work, no mention was made of the effect of 
different pressures at inoculation on virus titer and 
yield. To better understand the host pathogen inter-
action, data on MDMV titer in both susceptible and 
resistant cultivars is required. Enzyme-linked im-
munosorbent assay (ELISA) has been used to quan-
tify virus (2, 3, 10, 11, 12, 13, 14) and is a promising 
alternate to infectivity assay to study MDMV titer. 
The objeGtives of this study were to evaluate the 
relationship: among inoculation pressure virus titer, 
disease incidence, and disease severity in sorghum 
cultivars differing in reaction to MDMV-A. 
Materials and Methods 
Two sorghum cultivars, RTx430 (tolerant) and 
BTx378 (susceptible) were planted in randomized 
block designs replicated four times. Experimental 
? 
units were single rows, 6 meters long. Each cultivar 
was subjected to six different pressures (Table 1). 
Table 1. Air-gun pressures used for mechanical inocu-
lation of sorghum with MDMV-A. 
Treatment number Description 
1 not inoculated (control) 
2 1.76 kg/cm2 (25 Ib/sq inch) 
3 3.50 kg/cm2 (50 Ib/sq inch) 
4 5.28 kg/cm2 (75 Ib/sq inch) 
5 7.06 kg/cm2 (100 Ib/sq inch) 
6 8.81 ' kg/cm2 (120 Ib/sq inch) 
The different air pressures were provided to the 
spray gun by a portable 3000 watt (4Hp) gasoline 
powered air compressor. 
Inoculum Source 
MDMV - infected Johnsongrass (Sorghum he-
lepense) was used as the virus source. Infected 
Johnsongrass leaves were combined with 0.1M 
(mol/litre) potassium phosphate buffer, pH 7.2, at a 
1:4 (w/v) ratio and homogenized in a Waring blender. 
The resulting extract was expressed through four 
layers of cheesecloth. All operations were carried out 
on ice. One percent by volume of 600 mesh carborun-
dum was added to the extract to insure wounding of 
the leaf epidermis during inoculation. The abrasive 
was kept in suspension by periodical shaking during 
inoculation. 
Inoculation Procedure 
Plants were inoculated at the three leaf stage using 
an artist airbrush (DeVillbis MGA spray gun). Plants 
were sprayed at a distance of approximately 5 cm 
with an inoculum flow rate of 20 mllminute. 
Disease incidence due to virus infection was re-
corded 14 days after inoculation and thereafter at 
weekly intervals until booting. Each plant in the plot 
was rated for disease severity on a 1 to 5 scale (Table 
2). 
Table 2. Maize dwarf mosaic virus strain-A disease 
rating system. 
Category Description 
1 .0 Symptomless or healthy plants 
2.0 Mild to moderate mosaic symptoms 
3.0 Strong to severe mosaic symptoms, yellow-
ing with possible stunting. 
4.0 Slight necrotic reaction (red leaf response), 
strong to severe mosaic symptoms, usually 
with stunting. 
4.5 Necrosis over 20 to 50% of the leaves sur-
face area and growing tip. 
5.0 Necrosis over 50% of the leaves surface 
area and growing pOint, little or no useable 
ield ex ected, or dead lant. 
v 
A disease severity index (DS!) was calculated 
using the following equation: 
DSI = (a2 + b3 + ------ e5)/N 
where a, b, ------ e represent the number of infected 
plants within each symptom severity category and N 
is the total number of infected plants. 
Data also were collected for percent infection and 
yield and analyzed by ANOVA and regression. 
Tukey's studentised range test was used for mean 
separations. 
Enzyme-linked Immunosorbent Assay (ELISA) 
Titer of MDMV -A was assessed using the double 
antibody sandwich (DAS)-ELISA. Half leaf samples 
from the top-most fully expanded leaf from each of 10 
tagged plants in a plot were collected and combined. 
Plant tissue was macerated in plastic sleeves using a 
hammer and the sap was expressed with a Bantta-
ris's device and diluted 1:10 in O.lM potassium phos-
phate buffered saline solution (PBS) pH 7.2. 
Microliter plates were coated with 100 fll ofMDMV-A 
immunoglobin (lgG) and incubated overnight at 4 C. 
Coating of microliter plates with IgG, adding of 
antigen, biotinylated secondary antibody (conjugate), 
enzyme and substrate were done as described by 
Clark and Adams (1). Antigens (100 fll), including 
known concentrations of purified virus, buffer, and 
healthy control were added to ELISA plant wells 
according to a randomized block design with four 
replications. Antibody-antigen reactions were as-
sessed by adding 50 III of p-nitrophenyl phosphate 
substrate and incubating for 30 minutes at room 
temperature in the dark. Reactions were stopped by 
adding 50 III of 3M sodium hydroxide to each well. 
The absorbency of each sample at 410 nm was read 
on a Minireader II (Dynatech Laboratories, Alexan-
dria, VA), ELISA plate reader. 
Virus concentration, measured as optical density 
(aD) values, was analyzed by regression and 
ANOVA, and paired comparisons of treatment means 
within and between cultivars were done according to 
Tukey's studentised range test. 
Results 
The relationship between percentage infection and 
mechanical pressure used for inoculating MDMV-A 
was best described by a quadratic equation. For 
RTx430, the equation was: 
Y = 26.576 + 1.146 Xl - 0.0074 X2 
with R2 = 0.67 and Pr >F = 0.0001 
For Btx378 
Y = 38.911 + 0.823 Xl - 0.--5 X2 
with R2 = 0.18 and Pr >0.0001 
where Y = observed percentage infection 
Xl = inoculation pressure 
X2 = inoculation pressure squared 
All parameter estimates were highly significant 
with p < 0.0001. 
3 
Disease incidence was positively correlated (r = 
0.58 and r = 0.53 for RTx430 and BTx378, respec-
tively) with mechanical pressure used for inoculation. 
Paired comparisons of mean percentage infections 
showed no difference for inoculation pressures 
greater than 5.28 kg/cm2 for both RTx430 and 
BTx378. All inoculations resulted in significantly 
higher percentage infection compared to the non-in-
oculated controls (Table 3). 
Table 3. Mean percent infection of two sorghum culti-
vars following use of different inoculation pressures. 
Inoculation pressure Cultivar 
(kg/cm2) RTx430 BTx378 
0.00 28.11c 30.92b 
1.76 47.14bC 67.36a 
3.50 66.17ba 66.89a 
5.28 74.38a 68.92a 
7.06 64.46ba 65.40a 
8.81 58.47b 70.82a 
Fvalue 8.03 6.33 
Pr> F 0.0004 0.0017 
STD 11.699 10.714 
R2 0.69 0.65 
' . 
Least squares regression for disease severity index 
against inoculation pressure used were not signifi-
cant for either cultivar, and no differences were ob-
served for DSI among inoculation pressures (Figure 
1). There was no interaction between inoculation 
>< W 
C 
Z 
> !:: 
a: 
w 
> w 
en 
w 
en 
<t 
w 
~ 
c 
2.8~----------------------------------------------------------------------~ 
o 2 4 6 8 10 
INOCULATION PRESSURE Kg/Cm2 
Figure 1. Relationship between disease severity and inoculation 
pressure used for inoculating maize dwarf mosaic virus strain - A in 
two sorghum cultivars. 
'I 
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~I11II111111111 
A14838291828 
pressure and cultivar for disease severity and per-
centage infection. No correlations were observed 
among inoculation pressure, disease severity, and 
yield. 
ELISA 
Differences were observed for mean ELISA values 
for the different inoculation pressures used at inocu-
lation for RTx430. For BTx378, no differences were 
observed for ELISA values among the different pres-
sures used for inoculation. All inoculated plots had 
significantly higher mean ELISA values compared to 
the non-inoculated plots (Tables 4, 5). A high coeffi-
cient of determination (R = 0.96) was obtained be-
tween OD values and known concentrations of 
purified MDMV-A (Figure 2). Significant correlations 
(r = 0.60 for RTx430 and BTx378, respectively) were 
obtained between inoculation pressure and OD val-
ues for field data (Table 6). However, low nonsignifi-
cant correlations (r = 0.145 and 0.246 for RTx430 and 
BTx378, respectively) were obtained between OD and 
mechanical inoculation pressure for greenhouse data 
(Table 7). 
Table 4. Mean ELISA-values (00410) following use of 
different inoculation pressures in the field. 
Inoculation pressure Cultivar 
(kg/cm2) RTx430 BTx378 
0.00 0.28b 0.28b 
1.76 0.34ba OA3a 
3.50 0.30ba 0.44a 
5.28 0.39a 0.35a 
7.06 0.36ba 0.38a 
8.81 0.31 ba OA3a 
Healthy 0.02 0.02 
F value 2.18 3.93 
Pr> F 0.041 0.0035 
STO 0.0876 0.1127 
R2 0.18 0.23 
Table 5. Mean ELISA values (00410) of RTx430 and 
BTx378 following different inoculation pressures in the 
greenhouse. 
Inoculation pressure Cultivar 
(kg/cm2) RTx43 0 BTx378 
0.00 0.014b 0.014b 
1.76 0.340a OAOOa 
3.50 0.374a 0.388a 
5.28 0.382a OA12a 
7.06 ;i 0.393a OA02a 
8.81 0.388a OAOOa 
Healthy 0.010 0.010 
F value 52.37 66.02 
Pr> F 0.0001 0.0001 
STO 0.0847 0.07531 
R2 0.73 0.80 
4 
O.8r------------~,----, 
i= 0.6 
en 
Z 
w 
c 
<J. 0.4 
o 
i= Q. 
o 
0.2 
o.o~-~--~-~--~-~~-~ 
o 1 2 3 
LOG VIRUS CONC. 
Figure 2. Maize dwarf mosaic virus strain A standard curve. 
Table 6. Correlation coefficients (r) for data obtained 
from field stUdies. 
Variable RTx430 BTx378 
Pre x 00 0.308 0.391 
Pre x OSI 0.168 0.120 
OSI xOO 0.024 0.059 
INFxOO 0.160 0.598** 
INF x Pre 0.582** 0.527** 
Pre - pressure at inoculation 
00 - optical density (& ELISA) 
OSI- disease severity index 
INF - % infection 
** - significance at p < 0.001 
Table 7. Correlation coefficients (r) for data obtained 
from greenhouse studies. 
Variable RTx430 BTx378 
Pre x 00 0.732*** 0.668*** 
Pre x OSI 0.682*** 0.810*** 
OSI xOO 0.868*** 0.781*** 
INF x 00 0.904*** 0.948*** 
Pre x INF 0.732*** 0.712*** 
Pre - pressure at inoculation 
00 - optical density 
OSI- disease severity index 
INF - % infection 
*** - significance at p < 0.0001 
There was no association between virus titer and 
mechanical inoculation pressure (Figure 3). 
0.5 
0.4 
> t:: 
en 0.3 
z 
w 
C 
..J 
< o 0.2 
i= 
D.. 
o 
0.1 
D RT x 430 
[£] BT x 378 
o 1.76 3.5 5.28 7.06 8.81 
INOCULATION PRESSURE (Kg/Cm2) 
Figure 3. Mean ELISA values (OD410) obtained following use of 
different pressures when inoculating with MDMV-A (Field). 
Discussion 
Disease severity varied with mechanical inocula-
tion pressure. Toler and Miller (15) reported that 
"cultivars responded uniformly to inoculation pres-
sure and that rankings of cultivars by DSI remain 
unchanged". Results from this study show that there 
is no correlation between inoculation pressure and 
disease severity, but symptom expression was de-
layed when using pressures lower than 3.50 kg/cm2. 
Positive correlation (r = 0.58 and r = 0.53 for 
RTx430 and BTx378",respectively) were observed be-
tween percentage of infection and inoculation pres-
sure. The relationship was best described by a 
quadratic equation. However, pressures above 7.06 
kg/cm2 were associated with somewhat reduced dis-
ease incidence. This could be a result of lethal-cell 
damage by the high pressures during inoculation, 
which resulted in necrotic tissues and thereby imped-
ing virus multiplication and spread. For successful 
virus infection, non·lethal wounding of the leaf cells 
is essential. 
Weak negative correlations were obtained between 
inoculation pressure and yield. The weak associa-
tions could have resulted from the effect of the MDMV 
tolerant RTx430 plants that were asymptomatic and 
the high yielding capability of BTx378 plants that 
escaped infection. Titer, symptoms severity, yield, 
and pressure used at inoculation were not associated 
within the susceptible cultivar BTx378. 
Virus concentrations estimated by DAS-ELISA 
were not different among inoculation pressures be-
tween cultivars. Seifers and Hackerott (12), observed 
high virus concentrations in cultivars having red leaf 
reaction using MDMV-B. Giorda (2, 3), working with 
MDMV-A, obtained highest titer in BTx378, a suscep-
tible cultivar with necrotic reaction; the present 
study confirms these observations. The low correla-
tion obtained between OD values and air-gun pres-
sure at inoculation could be a result of natural 
infection by aphids. 
Low virus concentrations that were observed for 
the susceptible cultivar BTx378 compared to RTx430 
(mosaic reactor) might be due to the age of the tissue 
used for assaying virus titer. Seifers and Caceres (13) 
found that the sorghum cultivar Colt, which was 
resistant to MDMV-B, had lower ELISA values at 7 
days post-inoculation but not at 14, 21, and 28 days 
post-inoculation. Giorda (2,3) found that RTx430 had 
53 to 74 percent less MDMV-A accumulated than the 
susceptible cultivars RTx378, BTx3197 and RTx7000 
within the first 10 days after inoculation. This pro-
portion decreased thereafter depending on plant age 
and temperature. In this study, averages of three 
assessment times were used and this could have 
masked the differences between the two cultivars. 
BTx378 was observed to have numerous necrotic 
specks on the leaves and this could have impeded 
virus multiplication and reduced virus titer in the 
leaf, making titer variation with inoculation pressure 
difficult to ascertain. 
Literature Cited 
1. Clark, M. F., and Adams, A. N. 1977. Charac-
teristics of the microplate method of enzyme-
linked immunosorbent assay for the detection 
of plant viruses. J. Gen. Virol. 34:475-483. 
2. Giroda, L. M., Toler, R. W., and Miller, F. R. 1987. 
Concentration of maize dwarf mosaic virus 
strain A in sorghum cultivars with different 
reaction to the virus. Sorghum NL 30:83-87. 
3. Giorda, L. M., Toler, R. W., and Miller, F. R. 1987. 
Virus concentration in sorghum cultivars in-
oculated at different growth stages with maize 
dwarf mosaic virus strain A under greenhouse 
conditions. Sorghum NL 30:87-89. 
4. Law, M. D., Moyer, J. W., and Payne, G. A. 1989. 
Effects of host resistance on pathogenesis of 
maize dwarf mosaic virus. Phytopathology 
79:757-761. 
5. Louie, R., Findley, W. R., and Knoke, J. K. 1976. 
Variation in resistance within corn inbred 
lines to infection by maize dwarf mosaic virus. 
Plant Disease Reporter 60:838-842. 
6. Louie, R. 1986. Effects of genotype and inoculation 
protocols on resistance evaluation of maize 
dwarf mosaic virus strains. Phytopathology 
76:769-773. 
7. Mackenzie, D. R., Anderson, P. M., and Wernham, 
C. C. 1966. A mobile airblast inoculator for pot 
experiments with maize dwarf mosaic virus. 
Plant Disease Reporter 60:363-367. 
8. Martin, T. J., and Hackerott, H. I. 1982. Green-
house seedling technique to determine the re-
action of sorghum to maize dwarf mosaic virus 
strain A. Crop Science 22: 1255-1256. 
9. Miller, F. R., and Toler, R. W. 1977. Maize dwarf 
mosaic virus concentration, inoculation pres-
sure and variety interactions. Pages 87-97 in: 
Development of improved High-yielding Sor-
ghum Cultivars. 3rd Ann. Rept. USAID Tex 
Agric. Exp. Sta., College Station, TX 77843. 
10. Pataky, J. K., Murphy, J. F., and D'Arcy, C. J. 
1990. Resistance to maize dwarf mosaic virus, 
severity of symptoms, titer of virus, and yield 
of sweet corn. Plant Disease 74:359-364. 
11. Reeves, J. T., Jackson, A. 0., Paschke, J. D., and 
Lister, R. M. 1978. Use of enzyme-linked im-
munosorbent assay (ELISA) for serodiagnosis 
of two maize viruses. Plant Disease Reporter 
62:667- 671. 
12. Seifers, D. L., and Hackerott, H. L. 1987. Esti-
mates of yield loss and virus titre in sorghum 
hybrids infected with maize dwarf mosaic 
virus strain B. Agric. Ecosystems and Environ-
ment 19:81-86. 
13. Seifers, D. L., and Caceres, J. 1988. Titer variation 
in infected sorghum differing in resistance to 
maize dwarf mosaic virus strain B. Phytopa-
thology 78:208-212. 
14. Skaria, M., Lister, R. M., Foster, J. E., and 
Shaner, G. 1985. Virus content as an index of 
symptomatic resistance to barley yellow dwarf 
mosaic virus in cereals. Phytopathology 
75:212-216. 
15. Toler, R. W., and Miller, F. R. 1983. Effects of 
various combinations of inoculation pressure 
and concentration on varietal disease response 
of sorghum following spray gun inoculation 
with maize dwarf mosaic virus. Crop Science 
23:83-85. 
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English

Effect of Inoculation Pressure on Maize Dwarf Mosaic Virus Strain-A Disease Incidence, Severity and Titer in Sorghum.

Texas A&amp;M Repository

Z TA24S.7 8873 :--------------,1 NO.1706 8-1706 May 1992 TEXAS A&M UNIVERSITY LIBRARY Effect of Inoculation Pressure . on Maize Dwarf Mosaic Virus Strain-A Disease Incidence, Severity and Titer in Sorghum The Texas Agricultural Experiment Station • J. Charles Lee, Interim Director The Texas A&M University System • College Station, Texas ~ • . k ( :_ ., ., (Blank Page in OrigiBatBuUetiol I .I ,. : ::; , :" Effect of Inoculation Pressure on Maize Dwarf Mosaic Virus Strain-A Disease Incidence, Severity and Titer in Sorghum George s. Mahuku and R. W. Toler* * Plant Protection Research Institute, Department of Research and Specialist Services, P.O. Box 8100, Causeway, Harare, Zimbabwe, and Professor, Department of Plant Pathology and Microbiology, Texas Agricultural Experiment Station, Texas A&M University, College Station, TX 77843 ." Abstract Use of a spray gun for mechanical inoculation of viruses could result in different pressures during inoculation, affecting disease ratings of two sorghum cultivars. This experiment evaluated the effect of mechanical inoculation pressure on virus titer as measured by double antibody sandwich method of enzyme-linked immunosorbent assay (DAS-ELISA), disease incidence and severity in sorghum cultivars differing in reaction to maize dwarf mosaic virus strain-A (MDMV-A). Positive correlations (r=0.58 and r=.0.53 for RTx430 and BTx378 respectively), were obtained between disease incidence (percentage infection) when inoculation pressure was in the range of 5.28 to 7.06 kg/cm2. Neither disease severity nor titer was correlated to inoculation pressure. How-ever, inoculations resulted in higher virus titer com-pared to uninoculated plants. Introduction Maize dwarf mosaic virus (MDMV) occurs world wide in gramineous hosts and causes significant economic loss in several crops, including grain and forage sorghum, millet, and maize. Host plant resis-tance has reduced the losses caused by this virus disease. Artificial inoculation (using an airgun) of sorghum with MDMV has led to the release of tolerant and resistant inbreds and hybrids. When a spray gun is used for mechanical inoculation, differences in pres-sures during inoculation may result in selectively affected disease ratings among cultivars. Different field inoculation techniques have been used (6, 7,8, 9), and there is no consistency in the protocol fol-lowed. Inoculation pressures ranging from 4 kg/cm2 to 8.5 kg/cm2 (6, 8) have been used for field inocula-tions. There has been no mention of the effect of inoculation pressure on virus titer and yield. Toler and Miller (15), using four different inoculation pres-sures (0, 1.4, 4.2, and 7.3 kg. cm2), found increased cultivar susceptibility with increased pressure, and disease severity varied under different pressures. In their work, no mention was made of the effect of different pressures at inoculation on virus titer and yield. To better understand the host pathogen inter-action, data on MDMV titer in both susceptible and resistant cultivars is required. Enzyme-linked im-munosorbent assay (ELISA) has been used to quan-tify virus (2, 3, 10, 11, 12, 13, 14) and is a promising alternate to infectivity assay to study MDMV titer. The objeGtives of this study were to evaluate the relationship: among inoculation pressure virus titer, disease incidence, and disease severity in sorghum cultivars differing in reaction to MDMV-A. Materials and Methods Two sorghum cultivars, RTx430 (tolerant) and BTx378 (susceptible) were planted in randomized block designs replicated four times. Experimental ? units were single rows, 6 meters long. Each cultivar was subjected to six different pressures (Table 1). Table 1. Air-gun pressures used for mechanical inocu-lation of sorghum with MDMV-A. Treatment number Description 1 not inoculated (control) 2 1.76 kg/cm2 (25 Ib/sq inch) 3 3.50 kg/cm2 (50 Ib/sq inch) 4 5.28 kg/cm2 (75 Ib/sq inch) 5 7.06 kg/cm2 (100 Ib/sq inch) 6 8.81 ' kg/cm2 (120 Ib/sq inch) The different air pressures were provided to the spray gun by a portable 3000 watt (4Hp) gasoline powered air compressor. Inoculum Source MDMV - infected Johnsongrass (Sorghum he-lepense) was used as the virus source. Infected Johnsongrass leaves were combined with 0.1M (mol/litre) potassium phosphate buffer, pH 7.2, at a 1:4 (w/v) ratio and homogenized in a Waring blender. The resulting extract was expressed through four layers of cheesecloth. All operations were carried out on ice. One percent by volume of 600 mesh carborun-dum was added to the extract to insure wounding of the leaf epidermis during inoculation. The abrasive was kept in suspension by periodical shaking during inoculation. Inoculation Procedure Plants were inoculated at the three leaf stage using an artist airbrush (DeVillbis MGA spray gun). Plants were sprayed at a distance of approximately 5 cm with an inoculum flow rate of 20 mllminute. Disease incidence due to virus infection was re-corded 14 days after inoculation and thereafter at weekly intervals until booting. Each plant in the plot was rated for disease severity on a 1 to 5 scale (Table 2). Table 2. Maize dwarf mosaic virus strain-A disease rating system. Category Description 1 .0 Symptomless or healthy plants 2.0 Mild to moderate mosaic symptoms 3.0 Strong to severe mosaic symptoms, yellow-ing with possible stunting. 4.0 Slight necrotic reaction (red leaf response), strong to severe mosaic symptoms, usually with stunting. 4.5 Necrosis over 20 to 50% of the leaves sur-face area and growing tip. 5.0 Necrosis over 50% of the leaves surface area and growing pOint, little or no useable ield ex ected, or dead lant. v A disease severity index (DS!) was calculated using the following equation: DSI = (a2 + b3 + ------ e5)/N where a, b, ------ e represent the number of infected plants within each symptom severity category and N is the total number of infected plants. Data also were collected for percent infection and yield and analyzed by ANOVA and regression. Tukey's studentised range test was used for mean separations. Enzyme-linked Immunosorbent Assay (ELISA) Titer of MDMV -A was assessed using the double antibody sandwich (DAS)-ELISA. Half leaf samples from the top-most fully expanded leaf from each of 10 tagged plants in a plot were collected and combined. Plant tissue was macerated in plastic sleeves using a hammer and the sap was expressed with a Bantta-ris's device and diluted 1:10 in O.lM potassium phos-phate buffered saline solution (PBS) pH 7.2. Microliter plates were coated with 100 fll ofMDMV-A immunoglobin (lgG) and incubated overnight at 4 C. Coating of microliter plates with IgG, adding of antigen, biotinylated secondary antibody (conjugate), enzyme and substrate were done as described by Clark and Adams (1). Antigens (100 fll), including known concentrations of purified virus, buffer, and healthy control were added to ELISA plant wells according to a randomized block design with four replications. Antibody-antigen reactions were as-sessed by adding 50 III of p-nitrophenyl phosphate substrate and incubating for 30 minutes at room temperature in the dark. Reactions were stopped by adding 50 III of 3M sodium hydroxide to each well. The absorbency of each sample at 410 nm was read on a Minireader II (Dynatech Laboratories, Alexan-dria, VA), ELISA plate reader. Virus concentration, measured as optical density (aD) values, was analyzed by regression and ANOVA, and paired comparisons of treatment means within and between cultivars were done according to Tukey's studentised range test. Results The relationship between percentage infection and mechanical pressure used for inoculating MDMV-A was best described by a quadratic equation. For RTx430, the equation was: Y = 26.576 + 1.146 Xl - 0.0074 X2 with R2 = 0.67 and Pr >F = 0.0001 For Btx378 Y = 38.911 + 0.823 Xl - 0.--5 X2 with R2 = 0.18 and Pr >0.0001 where Y = observed percentage infection Xl = inoculation pressure X2 = inoculation pressure squared All parameter estimates were highly significant with p < 0.0001. 3 Disease incidence was positively correlated (r = 0.58 and r = 0.53 for RTx430 and BTx378, respec-tively) with mechanical pressure used for inoculation. Paired comparisons of mean percentage infections showed no difference for inoculation pressures greater than 5.28 kg/cm2 for both RTx430 and BTx378. All inoculations resulted in significantly higher percentage infection compared to the non-in-oculated controls (Table 3). Table 3. Mean percent infection of two sorghum culti-vars following use of different inoculation pressures. Inoculation pressure Cultivar (kg/cm2) RTx430 BTx378 0.00 28.11c 30.92b 1.76 47.14bC 67.36a 3.50 66.17ba 66.89a 5.28 74.38a 68.92a 7.06 64.46ba 65.40a 8.81 58.47b 70.82a Fvalue 8.03 6.33 Pr> F 0.0004 0.0017 STD 11.699 10.714 R2 0.69 0.65 ' . Least squares regression for disease severity index against inoculation pressure used were not signifi-cant for either cultivar, and no differences were ob-served for DSI among inoculation pressures (Figure 1). There was no interaction between inoculation >< W C Z > !:: a: w > w en w en <t w ~ c 2.8~----------------------------------------------------------------------~ o 2 4 6 8 10 INOCULATION PRESSURE Kg/Cm2 Figure 1. Relationship between disease severity and inoculation pressure used for inoculating maize dwarf mosaic virus strain - A in two sorghum cultivars. 'I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~I11II111111111 A14838291828 pressure and cultivar for disease severity and per-centage infection. No correlations were observed among inoculation pressure, disease severity, and yield. ELISA Differences were observed for mean ELISA values for the different inoculation pressures used at inocu-lation for RTx430. For BTx378, no differences were observed for ELISA values among the different pres-sures used for inoculation. All inoculated plots had significantly higher mean ELISA values compared to the non-inoculated plots (Tables 4, 5). A high coeffi-cient of determination (R = 0.96) was obtained be-tween OD values and known concentrations of purified MDMV-A (Figure 2). Significant correlations (r = 0.60 for RTx430 and BTx378, respectively) were obtained between inoculation pressure and OD val-ues for field data (Table 6). However, low nonsignifi-cant correlations (r = 0.145 and 0.246 for RTx430 and BTx378, respectively) were obtained between OD and mechanical inoculation pressure for greenhouse data (Table 7). Table 4. Mean ELISA-values (00410) following use of different inoculation pressures in the field. Inoculation pressure Cultivar (kg/cm2) RTx430 BTx378 0.00 0.28b 0.28b 1.76 0.34ba OA3a 3.50 0.30ba 0.44a 5.28 0.39a 0.35a 7.06 0.36ba 0.38a 8.81 0.31 ba OA3a Healthy 0.02 0.02 F value 2.18 3.93 Pr> F 0.041 0.0035 STO 0.0876 0.1127 R2 0.18 0.23 Table 5. Mean ELISA values (00410) of RTx430 and BTx378 following different inoculation pressures in the greenhouse. Inoculation pressure Cultivar (kg/cm2) RTx43 0 BTx378 0.00 0.014b 0.014b 1.76 0.340a OAOOa 3.50 0.374a 0.388a 5.28 0.382a OA12a 7.06 ;i 0.393a OA02a 8.81 0.388a OAOOa Healthy 0.010 0.010 F value 52.37 66.02 Pr> F 0.0001 0.0001 STO 0.0847 0.07531 R2 0.73 0.80 4 O.8r------------~,----, i= 0.6 en Z w c <J. 0.4 o i= Q. o 0.2 o.o~-~--~-~--~-~~-~ o 1 2 3 LOG VIRUS CONC. Figure 2. Maize dwarf mosaic virus strain A standard curve. Table 6. Correlation coefficients (r) for data obtained from field stUdies. Variable RTx430 BTx378 Pre x 00 0.308 0.391 Pre x OSI 0.168 0.120 OSI xOO 0.024 0.059 INFxOO 0.160 0.598** INF x Pre 0.582** 0.527** Pre - pressure at inoculation 00 - optical density (& ELISA) OSI- disease severity index INF - % infection ** - significance at p < 0.001 Table 7. Correlation coefficients (r) for data obtained from greenhouse studies. Variable RTx430 BTx378 Pre x 00 0.732*** 0.668*** Pre x OSI 0.682*** 0.810*** OSI xOO 0.868*** 0.781*** INF x 00 0.904*** 0.948*** Pre x INF 0.732*** 0.712*** Pre - pressure at inoculation 00 - optical density OSI- disease severity index INF - % infection *** - significance at p < 0.0001 There was no association between virus titer and mechanical inoculation pressure (Figure 3). 0.5 0.4 > t:: en 0.3 z w C ..J < o 0.2 i= D.. o 0.1 D RT x 430 [£] BT x 378 o 1.76 3.5 5.28 7.06 8.81 INOCULATION PRESSURE (Kg/Cm2) Figure 3. Mean ELISA values (OD410) obtained following use of different pressures when inoculating with MDMV-A (Field). Discussion Disease severity varied with mechanical inocula-tion pressure. Toler and Miller (15) reported that "cultivars responded uniformly to inoculation pres-sure and that rankings of cultivars by DSI remain unchanged". Results from this study show that there is no correlation between inoculation pressure and disease severity, but symptom expression was de-layed when using pressures lower than 3.50 kg/cm2. Positive correlation (r = 0.58 and r = 0.53 for RTx430 and BTx378",respectively) were observed be-tween percentage of infection and inoculation pres-sure. The relationship was best described by a quadratic equation. However, pressures above 7.06 kg/cm2 were associated with somewhat reduced dis-ease incidence. This could be a result of lethal-cell damage by the high pressures during inoculation, which resulted in necrotic tissues and thereby imped-ing virus multiplication and spread. For successful virus infection, non·lethal wounding of the leaf cells is essential. Weak negative correlations were obtained between inoculation pressure and yield. The weak associa-tions could have resulted from the effect of the MDMV tolerant RTx430 plants that were asymptomatic and the high yielding capability of BTx378 plants that escaped infection. Titer, symptoms severity, yield, and pressure used at inoculation were not associated within the susceptible cultivar BTx378. Virus concentrations estimated by DAS-ELISA were not different among inoculation pressures be-tween cultivars. Seifers and Hackerott (12), observed high virus concentrations in cultivars having red leaf reaction using MDMV-B. Giorda (2, 3), working with MDMV-A, obtained highest titer in BTx378, a suscep-tible cultivar with necrotic reaction; the present study confirms these observations. The low correla-tion obtained between OD values and air-gun pres-sure at inoculation could be a result of natural infection by aphids. Low virus concentrations that were observed for the susceptible cultivar BTx378 compared to RTx430 (mosaic reactor) might be due to the age of the tissue used for assaying virus titer. Seifers and Caceres (13) found that the sorghum cultivar Colt, which was resistant to MDMV-B, had lower ELISA values at 7 days post-inoculation but not at 14, 21, and 28 days post-inoculation. Giorda (2,3) found that RTx430 had 53 to 74 percent less MDMV-A accumulated than the susceptible cultivars RTx378, BTx3197 and RTx7000 within the first 10 days after inoculation. This pro-portion decreased thereafter depending on plant age and temperature. In this study, averages of three assessment times were used and this could have masked the differences between the two cultivars. BTx378 was observed to have numerous necrotic specks on the leaves and this could have impeded virus multiplication and reduced virus titer in the leaf, making titer variation with inoculation pressure difficult to ascertain. Literature Cited 1. Clark, M. F., and Adams, A. N. 1977. Charac-teristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34:475-483. 2. Giroda, L. M., Toler, R. W., and Miller, F. R. 1987. Concentration of maize dwarf mosaic virus strain A in sorghum cultivars with different reaction to the virus. Sorghum NL 30:83-87. 3. Giorda, L. M., Toler, R. W., and Miller, F. R. 1987. Virus concentration in sorghum cultivars in-oculated at different growth stages with maize dwarf mosaic virus strain A under greenhouse conditions. Sorghum NL 30:87-89. 4. Law, M. D., Moyer, J. W., and Payne, G. A. 1989. Effects of host resistance on pathogenesis of maize dwarf mosaic virus. Phytopathology 79:757-761. 5. Louie, R., Findley, W. R., and Knoke, J. K. 1976. Variation in resistance within corn inbred lines to infection by maize dwarf mosaic virus. Plant Disease Reporter 60:838-842. 6. Louie, R. 1986. Effects of genotype and inoculation protocols on resistance evaluation of maize dwarf mosaic virus strains. Phytopathology 76:769-773. 7. Mackenzie, D. R., Anderson, P. M., and Wernham, C. C. 1966. A mobile airblast inoculator for pot experiments with maize dwarf mosaic virus. Plant Disease Reporter 60:363-367. 8. Martin, T. J., and Hackerott, H. I. 1982. Green-house seedling technique to determine the re-action of sorghum to maize dwarf mosaic virus strain A. Crop Science 22: 1255-1256. 9. Miller, F. R., and Toler, R. W. 1977. Maize dwarf mosaic virus concentration, inoculation pres-sure and variety interactions. Pages 87-97 in: Development of improved High-yielding Sor-ghum Cultivars. 3rd Ann. Rept. USAID Tex Agric. Exp. Sta., College Station, TX 77843. 10. Pataky, J. K., Murphy, J. F., and D'Arcy, C. J. 1990. Resistance to maize dwarf mosaic virus, severity of symptoms, titer of virus, and yield of sweet corn. Plant Disease 74:359-364. 11. Reeves, J. T., Jackson, A. 0., Paschke, J. D., and Lister, R. M. 1978. Use of enzyme-linked im-munosorbent assay (ELISA) for serodiagnosis of two maize viruses. Plant Disease Reporter 62:667- 671. 12. Seifers, D. L., and Hackerott, H. L. 1987. Esti-mates of yield loss and virus titre in sorghum hybrids infected with maize dwarf mosaic virus strain B. Agric. Ecosystems and Environ-ment 19:81-86. 13. Seifers, D. L., and Caceres, J. 1988. Titer variation in infected sorghum differing in resistance to maize dwarf mosaic virus strain B. Phytopa-thology 78:208-212. 14. Skaria, M., Lister, R. M., Foster, J. E., and Shaner, G. 1985. Virus content as an index of symptomatic resistance to barley yellow dwarf mosaic virus in cereals. Phytopathology 75:212-216. 15. Toler, R. W., and Miller, F. R. 1983. Effects of various combinations of inoculation pressure and concentration on varietal disease response of sorghum following spray gun inoculation with maize dwarf mosaic virus. Crop Science 23:83-85. Mention of a trademark or a proprietary product does not constitute a guarantee or a warranty of the product by The Texas Agricultural Experiment Station and does not imply its approval to the exclusion of other products that also may be suitable. All programs and information of The Texas Agricultural Experiment Station are available to everyone without re-gard to race, color, religion, sex, age, handicap, or national origin. 

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Effect of Inoculation Pressure on Maize Dwarf Mosaic Virus Strain-A Disease Incidence, Severity and Titer in Sorghum.

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Effect of Inoculation Pressure on Maize Dwarf Mosaic Virus Strain-A Disease Incidence, Severity and Titer in Sorghum.

Abstract

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