ikfiihs]
B873
NO.1706 5-1706 May 1992

1y,‘

%

TEXAS A&M UNIVERSITY LIBRARY

Effect 0f 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

i. 

[Blank Page in Original Bulletin] A

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/cmz. 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/cm
to 8.5 kg/cmz (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. cmz), 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 objectives 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)
1.1a kg/cmz (25 lb/sq inch)
3.50 kg/cmz (50 lb/sq inch)
5.2a kg/cmz (75 lb/sq inch)
7.06 kg/cmz (100 lb/sq inch)

(DUI-kWh)

8.81 kg/cmz (120 lb/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 ml/minute.

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
yield expected, or dead plant.

‘T

9

Q

i"

A disease severity index (DSI) 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 0.1M potassium phos-
phate buffered saline solution (PBS) pH 7.2.
Microliter plates were coated with 100 pl of MDMV-A
immunoglobin (IgG) and incubated overnight at 4 C.

Coating of microliter plates with IgG, adding 0f
antigen, biotinylated secondary antibody (conjugate),
enzyme and substrate were done as described by
Clark and Adams (1). Antigens (100 p1), 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 pl of p-nitrophenyl phosphate
substrate and incubating for 30 minutes at room
temperature in the dark. Reactions were stopped by
adding 50 ul 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
(DD) values, was analyzed by regression and
AN OVA, 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 X1 - 0.0074 X2
with R2 = 0.67 and Pr >F = 0.0001

For Btx378

Y = 38.911 + 0.823 X1 - 0.--5 X2
with R2 = 0.18 and Pr >0.0001
where Y = observed percentage infection

X1 = inoculation pressure

X2 = inoculation pressure squared

All parameter estimates were highly significant
with p < 0.0001.

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/cmz 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/cmz) RTx43O BTx378
0.00 26.11° 60.92“

1 .76 47.14“ 67.66“
6.50 66.17“ 6669a
5.26 74.363 66.92‘
7.06 64.46” 65.40“
6.61 56.47” 7062a

F value 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
l). There was no interaction between inoculation

2.8

2.7

RTX430

2.6

   

BTX378

2.5

DISEASE SEVERITY INDEX

0 2 4 6 8 10

INOCULATION PRESSURE Kg/Cmz

Figure 1. Relationship between disease severity and inoculation
pressure used for inoculating maize dwarf mosaic virus strain - A in
two sorghum cultivars.

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 ELlSAvalues (CD410) following use of
different inoculation pressures in the field.
Inoculation pressure Cultivar

(kg/cmz) RTx430 BTx378
0.00 0.28“ 0.28“
1.76 0.34““ 0.43“
3.50 0.30““ 0.44“
5.28 0.393 0.35“
7.06 0.36““ 0.38“
8.81 0.31““ 0.43“
Healthy 0.02 0.02
Fvalue 2.18 3.93
Pr > F 0.041 0.0035
STD 0.0876 0.1127
R“ 0.18 0.23

Table 5. Mean ELISA values (CD410) 0f RTx430 and
BTx378 following different inoculation pressures in the
greenhouse.
Inoculation pressure Cultivar
(kg/cmz) RTx430 BTx378
0.00 0.014“ 0.014“
1.76 0.340“ 0.400“
3.50 0.374“ 0.388“
5.28 0.382“ 0.412“
7.06 _ 0.393“ 0.402“
8.81 ~ 0.388“ 0.400“
Healthy 0.010 0.010
F value 52.37 66.02
Pr > F 0.0001 0.0001
STD 0.0847 0.07531
R“ 0.73 0.80

OPTICAL DENSITY

0D l I I l I

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 OD 0.308 0.391
Pre X DSI 0.168 0.120
DSI x OD 0.024 0.059
lNF x OD 0.160 0.598**
lNF x Pre 0.582** 0.527“

Pre - pressure at inoculation
OD - optical density (& ELISA)
DSI - disease severity index
lNF - % infection

** - significance at p < 0.001

Table 7. Correlation coefficients (r) for data obtained
from greenhouse studies.

Variable RTx430 BTx378
Pre x CD 0.732*** 0.668***
Pre x DSI 0682*“ 0.810***
DSI x CD 0.868*** 0.781***
INF x CD 0.904*** 0.948***
Pre x INF 0732*“ O.712***

Pre - pressure at inoculation

CD - optical density

DSI - 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

D RT X 430

BT x 37a

 
    

 

OPTICAL DENSITY
P
l

              

0.0  - I
o 1.76
INOCULATION PRESSURE (Kg/Cmz)

Figure 3. Mean ELISA values (OD410) obtained following use of
different pressures when inoculating with MDMV-A (Field).

I 33'3"] v
28 7.06 8.81

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/cmz.

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/cm 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.

Mention of a trademark or a proprietary product does not constitute a guarantee or a warranty of the product by

12. Seifers, D. L., and Hackerott, H. L. 1987. Esti-
mates of yield loss and virus titre in sorghum
hybrids infected with maize dwarf mosair
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.

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.

/_