Aphid performance changes with plant defense mediated by Cucumber mosaic virus titer
Shi et al. Virology Journal
Aphid performance changes with plant defense mediated by Cucumber mosaic virus titer
Xiaobin Shi 1
Yang Gao 1
Shuo Yan 1
Xin Tang 1
Xuguo Zhou 2
Deyong Zhang 0 1
Yong Liu 0 1
0 Longping Branch, Graduate College, Hunan University , Changsha 410125 , China
1 Key Laboratory of Integrated Management of the Pests and Diseases on Horticultural Crops in Hunan Province, Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences , Changsha 410125 , China
2 Department of Entomology, University of Kentucky , Lexington, KY 40546 , USA
Background: Cucumber mosaic virus (CMV) causes appreciable losses in vegetables, ornamentals and agricultural crops. The green peach aphid, Myzus persicae Sulzer (Aphididae) is one of the most efficient vectors for CMV. The transmission ecology of aphid-vectored CMV has been well investigated. However, the detailed description of the dynamic change in the plant-CMV-aphid interaction associated with plant defense and virus epidemics is not well known. Results: In this report, we investigated the relationship of virus titer with plant defense of salicylic acid (SA) and jasmonic acid (JA) during the different infection time and their interaction with aphids in CMV-infected tobacco plants. Our results showed that aphid performance changed with virus titer and plant defense on CMV-inoculated plants. At first, plant defense was low and aphid number increased gradually. The plant defense of SA signaling pathway was induced when virus titer was at a high level, and aphid performance was correspondingly reduced. Additionally, the winged aphids were increased. Conclusion: Our results showed that aphid performance was reduced due to the induced plant defense mediated by Cucumber mosaic virus titer. Additionally, some wingless aphids became to winged aphids. In this way CMV could be transmitted with the migration of winged aphids. We should take measures to prevent aphids in the early stage of their occurrence in the field to prevent virus outbreak.
Cucumber mosaic virus; Myzus persicae; Plant defense; Jasmonic acid; Salicylic acid
Plants are constantly attacked by many kinds of plant
viruses, and they have evolved extraordinarily complex
mechanisms to defend themselves [
]. About 80 % plant
viruses depend on insect vectors for transmission [
At present, the triple plant-virus-vector interaction has
been paid more and more attention for understanding
the complex interplay of factors resulting in virus
emergence. In the current study, we investigate the triple
interactions between Cucumber mosaic virus (CMV),
Myzus persicae Sulzer (Aphididae), and tobacco plants.
Cucumber mosaic virus (CMV) causes appreciable
losses in vegetables, ornamentals and agricultural crops
]. CMV has a broad host range, including more than
1, 200 plant species in over 100 families [
]. CMV is
transmitted by 80 species of aphids in 33 genera in a
non-persistent manner [
]. The green peach aphid, M.
persicae is one of the most efficient vectors for CMV [
and is frequently used in transmission experiments [
Plants can antagonize the growth, development and
preference of insect vectors directly and therefore
affect virus transmission indirectly. The plant–vector
interaction appears favorable to the persistent
transmission, such as those described for Barley yellow
dwarf virus (BYDV) and Potato leaf roll virus (PLRV),
that both attract vectors to and encourage their
population growth and sustained feeding on infected plants
]. Our previous results also showed that
infection of Tomato yellow leaf curl virus (TYLCV)
increased the performance of whiteflies, Bemisia
tabaci to facilitate virus transmission [
Previous research, however, revealed the different pattern
of plant-vector interaction for transmission of
nonpersistent viruses from that of the transmission of
persistent viruses . CMV-infected squash plants
are poor hosts for aphid vectors, while aphids
exhibited a preference for the elevated volatile emissions of
infected plants. Besides, CMV infection induces changes
in host palatability and quality for aphid vectors rapid
dispersal following virus acquisition [
transmission ecology of aphid-vectored CMV has been well
investigated, and interactions between viruses and
aphids are key factors influencing CMV epidemics
. However, the detailed description of the dynamic
change in the plant-CMV-aphid interaction associated
with plant defense and virus epidemics was not well
The signaling pathways in this plant-CMV-aphid
interaction influence each other through a complex
network of synergistic and antagonistic interactions
]. The phytohormones salicylic acid (SA) and
jasmonic acid (JA) are known to participate in defense
responses in plants [
]. There is considerable
cross-talk between JA and SA [
]. In plant-insect
interactions, SA induction has been confirmed to be
an effective defense response against aphids and
]. β-1, 3-glucanases is an important
pathogenesis-related (PR) protein in response to
pathogenic infection mediated by SA (Livne [
]). Protease inhibitor is caused by JA as a
result of injury (Turner [
] Zhang et al. [
In the process of virus infection, whether the virus
titer has a time effect on plant defense such as JA
and SA in molecular and biochemical level which can
regulate aphid performance is largely unknown. In
this report, we investigated the relationship of virus
titer with SA and JA during the different infection
time and their interaction with virus vector aphids in
CMV-infected tobacco plants. Our goal was to find
some ecological mechanisms in the virus epidemics of
Plant, aphid colonies and virus culture
Tobacco plants (Nicotiana tabacum cv. Samsum) were
grown in a potting mix (a mixture of vermiculite, peat
moss, organic fertilizer and perlite in a 10:10:10:1 ratio
by volume) in insect-free cages (60 × 60 × 60 cm) in a
glasshouse. Myzus persicae (Sulzer) were raised in
colonies on tobacco plants. When plants were at the 3–4
true leaf stage, they were inoculated with 5 cm2 of frozen
stock tissue infected with CMV (stored at −80 °C).
Frozen tissue was ground with 5 ml of 0.1 M potassium
phosphate buffer on a cold surface. Carborundum
powder was then added and the mixture was applied to
surfaces of tobacco leaves using cotton swabs. Control
plants were mock-inoculated in the same manner, but
with healthy tobacco tissue.
Viral load with DAS-ELISA
Virus titer was determined after 3, 6, 9, 12 and 15 days
of CMV-inoculation. The identity of the virus titer was
detected though DAS-ELISA using diagnostic kit
JA and SA in molecular and biochemical level
The gene expression of the JA and SA signaling pathway
in CMV-inoculated plants were determined at 3, 9 and
15 days post-inoculation. The JA upstream gene OPR3
and downstream genes COI1 and PDF1.2 were
measured. At the same time the SA upstream gene ICS1 and
downstream genes NPR1 and PR1 were measured. Actin
was used as the internal reference gene [
]. Total RNA
was extracted from 0.2 g of CMV-inoculated leaves, and
1.0 μg of RNA was used to synthesize the first-strand
cDNA using the PrimeScript® RT reagent Kit (Takara
Bio, Tokyo, Japan) with gDNA Eraser (Perfect Real
Time, TaKara, Shiga, Japan). The 25.0 μl reaction system
containing 10.5 μl of ddH2O, 1.0 μl of cDNA, 12.5 μl of
SYBR® Green PCR Master Mix (TIANGEN, Beijing,
China), and 0.5 μl of each primer (Table 1). Relative
quantities of RNA were calculated using the comparative cycle
threshold (Ct) (2-ΔΔCt) method [
]. Three biological
replicates and four technical replicates were analyzed.
The activity of proteinase inhibitor (PI) and β-1,
3glucanases (GUS) of CMV-inoculated leaves were
determined at 3, 9 and 15 days post-inoculation. The activity
of PI was determined using standard protocol [
activity of GUS was determined using standard protocol
]. GUS activity was calculated as nmoles of MU per
minute per milligram of protein. Three biological
replicates and three technical replicates were analyzed.
Vaseline was plastered at the culm of the tobacco to
prevent aphids from escaping. After a 4 h equilibration
period, 20 apterous adults of the same age, which had
been starved for 4 h, were placed separately on
mockinoculated and CMV-infected tobacco plants. The
mock-inoculated and CMV-infected tobacco plants with
aphids were placed separately in insect-free cages (40 ×
20 × 40 cm).
After 3, 6, 9, 12 and 15 days, the number of apterous
and alate aphids on mock-inoculated and CMV-infected
plants were count and recorded. In order not to miss
any aphids, all the spaces in the cages were also checked.
After each count, winged aphids were all removed by an
aspirator, in order not to interfere the observation in the
next time. Each experiment was repeated eight times.
To determine the longevity, aphids was collected and
transferred to mock-inoculated and CMV-infected
tobacco plants. Each plant was placed 20 apterous adults
of the same age. The new born aphids were removed
and every female was checked every day until their
death, and the longevity of aphids was recorded.
One-way ANOVA was used to compare viral titer of
CMV-infected leaves and to compare relative gene
expression and enzyme activity of JA and SA signaling
pathway. One-way ANOVA was also used to compare
number of winged aphids on CMV-infected plants.
Repeated-measures ANOVAs were used to compare the
number of aphids on mock-inoculated plants and
CMVinfected plants. Longevity of aphids on mock-inoculated
plants and CMV-infected plants were compared with
Viral load with DAS-ELISA
Viral load differed significantly in the first 15 days (F =
17.462, P < 0.001). Virus titer grew continuously at the
beginning, and then, from the 9th day, virus titer
remained at a relatively stable level (Fig. 1).
JA and SA in molecular and biochemical level
The expression of the JA upstream gene OPR3 was
increased, and the expression of the JA downstream genes
COI1 and PDF1.2 was decreased. Besides, the expression
of COI1 and PDF1.2 was number numerically lowest on
the 9th day (Fig. 2a). The expression of SA upstream
gene ICS1 and downstream genes NPR1 and PR1 was
increased on CMV-inoculated plants. The SA-responsive
gene expression was numerically highest on the 9th day
PI activity was reduced from 3 days to 15 days and was
numerically lowest on the 9th day (one-way ANOVA: F =
15.023, P = 0.005, Fig. 2c). GUS activity was increased
from 3 days to 15 days and was numerically highest on
the 9th day (one-way ANOVA: F = 15.902, P = 0.004,
On the third day, aphid number was similar on
CMVinfected plants and mock-inoculated plants. From the
6th day to the 15th day, there was significant difference
in aphid numbers between individuals on CMV-infected
plants and mock-inoculated plants
(repeated-measurement ANOVA: F = 2739.310, P < 0.001). Aphid growth
rate on mock-inoculated plants only changed a little.
However, aphid growth rate on CMV-infected plants
slowed down gradually (Fig. 3a). The longevity of aphid on
CMV-infected plants was significantly lower than on
mock-inoculated plants (F = 0.293, P = 0.005; Fig. 3b).
The number of winged aphids increased on
CMVinfected plants from the 9th day, and the number of
winged aphids was highest on CMV-infected plants on
the 15th day (Fig. 3c).
Our results demonstrate that there is a clear link
between the aphid number, virus titer and plant defense.
The number of aphids on virus-infected plants
significantly increased in the beginning, but the number
declined significantly after the virus titer maintained a
certain level. Besides, the longevity of aphids on
CMVinfected plants was lower than that on mock-inoculated
plants. Our results show that in the early infection of
CMV, infected plants can promote the growth and
development of aphids. However, when the viral titer
remained stable in plants, the growth of aphid was
decreased. One previous result showed that survival of M.
persicae was lower on CMV-infected tobacco, as
compared to mock-inoculated plants within 14 days [
Another previous result showed that performance of M.
persicae was dramatically reduced on CMV-infected
plants within 15 days [
]. Combined with our results,
we can find that aphid performance on CMV-infected
plants is time-dependent.
Our results also showed that plant defense changed
with the increase of virus titer. In the initial stages of
CMV infection, only the expression of JA upstream gene
OPR3 was induced to a higher level. However, the
SArelative genes were induced a little. On the 9th day when
the viral titer was highest, the expression of
SAresponsive genes such as NPR1 was highest, and the
expression of JA downstream genes was lowest. NPR1 has
been reported to play a key role in the regulation of SA
and JA antagonism [
]. For example, the infection of
necrotrophic fungus Botrytis cinerea activates SA
signaling via a tomato NPR1 homolog to exploit the
antagonistic crosstalk between SA and JA signaling [
]. The 2b
protein of CMV targets NPR1 to exploit SA-JA
]. Our previous also showed that infection of
TYLCV induced the NPR1 expression and reduced the
JA downstream gene expression [
]. Here we found
that the NPR1 and PR1 both were induced by CMV
infection and they play important roles in the
antagonistic crosstalk between the SA and JA pathways. Therefore,
CMV infection induced SA-regulated gene expression and
disrupted JA-regulated gene expression, which is
consistent with previous results [
Proteinase inhibitor plays an important role in
resisting insect herbivores and has been reported to
be related with JA [
]. β-1, 3-glucanases is also an
important enzyme that is involved in response to
salicylic acid (SA) [
]. In our results PI activity was
reduced while GUS activity was increased, which is
consistent with the expression change of JA and SA
According to our results, compared with the
mockinoculated plants, plant defense on CMV-infected
plants in its early stage was low, and the aphid
number increased rapidly. However, when the viral titer
remained stable in plants, plant defense especially
SAresponsive genes were induced to a higher level. SA
can have neutral or negative effects on the growth of
]. Avila et al. [
] showed that FAD7
enhances plant defenses against aphids that are
mediated through SA and NPR1. SA induction has been
confirmed to be an effective chemical defense
response against aphids [
]. In our research SA were
induced by the increase of virus titer and therefore
the growth of aphid was decreased.
Another possibility to consider is that plant quality is
changed by infection of CMV. Previous research showed
that CMV infection reduces the host palatability and
quality, and the phloem sap quality is also reduced [
Combined with our results, it can be found that with the
increase of viral titer, aphid performance can be reduced
due to the reduction of plant physiology and
We also found that number of winged aphids
increased with the increase of viral titer. Many factors,
such as environmental conditions, aphid density and
host plant quality, may influence wing production [
For example, a decrease in plant quality can trigger wing
induction in some aphid species [
]. Here, we show
that winged M. persicae on virus-infected leaves are
more than on mock-inoculated leaves after 9 days,
although the number of aphids on virus-infected leaves is
lower than on mock-inoculated leaves. Therefore, we
consider it unlikely that our result is caused by aphid
density. The possible explanation is that plant defense is
induced by CMV infection therefore plant quality is
changed, which is consistent with previous results that
plant quality decreases under infection of non-persistent
viruses to promote aphid migration [
]. In our results
we found that plant quality decreases under infection of
non-persistent viruses and then the wingless aphids
become to winged aphids.
We find some ecological mechanisms in the virus
epidemics of CMV. The aphid performance changed with
virus titer and plant defense on CMV-inoculated plants.
At first, plant defense was low and aphid number
increased gradually. The plant defense of SA signaling
pathway was induced when virus titer was at a high
level, and aphid performance was correspondingly
reduced. Additionally, the wingless aphids became to
winged aphids. CMV could be transmitted with the
migration of winged aphids. We should take measures to
prevent aphids in the early stage of their occurrence in
the field to prevent virus outbreak. The physiological,
biochemical and molecular mechanisms of wing
production need to be further investigated.
The authors declare that they have no competing interests.
XBS, DYZ and YL designed the experiment. XBS, SY and XT carried the
experimental work. YG and XGZ contributed reagents/materials. XBS wrote
the paper. All authors read and approved the final manuscript.
This work was supported by the Special Fund for Agro-scientific Research in
the Public Interest (no. 201303028), the Agriculture Research System of China
(CARS-25-B-05) and the national natural science foundation of china (no.
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