Preference of cereal aphids for different varieties ... - Insects.ugent.be

Arthropod-Plant Interactions (2012) 6:345–350
DOI 10.1007/s11829-012-9184-5
ORIGINAL PAPER
Preference of cereal aphids for different varieties of winter wheat
Nathalie De Zutter • Kris Audenaert •
Geert Haesaert • Guy Smagghe
Received: 15 November 2011 / Accepted: 15 February 2012 / Published online: 3 March 2012
Ó Springer Science+Business Media B.V. 2012
Abstract This paper reports on the development of a
simple and robust preference meter (developed in-house) to
score the host choice behavior of apterous aphids. With this
tool, the preferences of two important cereal aphids Sitobion
avenae (Fab.) and Metopolophium dirhodum (Walker)
were investigated against four different varieties of winter
wheat (Triticum aestivum L.) with a different susceptibility
for Fusarium head blight (FHB). Differences in the choice
behavior of both aphid species were observed for different
wheat varieties. The preferred wheat variety of S. avenae
and M. dirhodum was not the same. Also, both aphid
species clearly had a differential preference for seedlings
and ears. Using seedlings, M. dirhodum was about 1.8
times more rapid in making its choice than S. avenae. In
separate experiments with ears, S. avenae was 4.5 times
faster than in the experiments with seedlings. In the present
study, we aim to highlight differences in preference
behavior in relation to potential mechanisms for host
selection.
Keywords Apterae Cereal aphids Metopolophium
dirhodum Sitobion avenae Variety preference Winter
wheat
Handling Editor: Robert Glinwood.
N. De Zutter G. Smagghe (&)
Department of Crop Protection, Faculty of Bioscience
Engineering, Ghent University, Coupure Links 653, 9000 Ghent,
Belgium
e-mail: guy.smagghe@ugent.be
N. De Zutter K. Audenaert G. Haesaert
Faculty of Applied Bioscience Engineering, University College
Ghent, Valentin Vaerwyckweg 1, 9000 Ghent, Belgium
Introduction
Wheat is one of the world’s most important food grains and
satisfies a considerable part of the human nutritional needs
(Bushuk 1998). In 2009, over 225 million hectares of
wheat were harvested which had a gross production value
of more than $145 billion (FAOSTAT 2011). Nowadays,
pre- and post-harvest infestations by insects are among the
major constraints in the production of wheat.
One of the major pests of wheat is the aphid (Hemiptera:
Aphididae). These insects damage crops by feeding on the
plant assimilates or by transmitting viruses. Two important
species of wheat in NW Europe are Sitobion avenae (Fab.)
(English grain aphid) and Metopolophium dirhodum
(Walker) (rose-grain aphid). Since S. avenae first feeds on
the leaves but later migrates to the ears after emergence,
they are known as ear feeders (Wratten 1975). M. dirhodum
prefers leaves and rarely moves to the ears (Nyaanga
et al. 2006). This leaf feeder migrates to the flag leaf once
the lower leaves start to senesce (Wratten 1975).
Preference assays are imperative to delineate the
importance of the host genotype in the behavior of aphids
when they have to choose between different host varieties.
It also contributes to a judicious selection of wheat varieties
in order to avoid or limit aphid infestations and thus
minimize yield losses.
Plant chemicals can play a role in the host selection of
aphids. These chemicals are part of a defensive network
protecting the plant from insect attack. For instance, Lushai
et al. (1997) investigated the host selection of winged summer
females of S. avenae. Their approach involved counting
the progeny in host preference tests, from which they concluded
that aphids preferred the agricultural host winter
wheat more than its natural host cocksfoot (Dactylis glomerata
L.). Although molecular mechanisms behind this
123

346 N. De Zutter et al.
preference largely remain elusive, Nicol et al. (1992) pointed
to a significant negative relationship between the proportion
of alate S. avenae colonizing seedlings of different wheat
varieties and the hydroxamic acid levels in those varieties.
There is also a reasonable likelihood that volatile
organic compounds (VOC) are important triggers for
choosing a host plant. Several studies corroborate the role
of plant volatiles in the attraction of aphids to plants. For
example, Quiroz and Niemeyer (1998) noticed an elicited
attraction of the cereal aphid Rhopalosiphum padi L. to
volatiles produced by wheat and oat seedlings. Although
plants produce volatiles during their physiological development,
biosynthesis of volatiles is especially triggered
when fungal or bacterial plant pathogens infect and colonize
plants (Cardoza et al. 2002; Huang et al. 2003; Obara
et al. 2002; Toome et al. 2010; Vuorinen et al. 2007).
The aphid preference also sheds light on the tritrophic
interaction between wheat, cereal aphids and wheat fungi.
Indeed, if cereal aphids like S. avenae and M. dirhodum prefer
certain wheat varieties that are also susceptible to plant
pathogenic fungi like Fusarium sp., both insects and fungi
could interact resulting in a faster dispersal of fungal mycelium
and spores. This has already been described for the
orange wheat blossom midge (Sitodiplosis mosellana
(Géhin)) and Fusarium graminearum (Schwabe) macroconidia
(Mongrain et al. 2000). They recovered the fungus from
the spikes of wheat plants that had been exposed to artificially
inoculated midges. On other plants, interactions between
insects and fungal plant pathogens have already been
described. For example, pea aphids Acyrthosiphon pisum
(Harris) are a vector of Verticillium albo-atrum (Reinke and
Berthold) on alfalfa (Medicago sativa L.) (Huang et al. 1981).
Adult shore flies, fungus gnats and moth flies are vectors of
Fusarium avenaceum (Fr.:Fr.) Sacc. that causes Fusarium
crown and stem rot on lisianthus (Eustoma grandiflorum Raf.
(Shinn.)) (El-Hamalawi and Stanghellini 2005).
In this paper, we report on an in-house developed
preference meter that is simple, robust and used to investigate
the preference behavior of apterous aphids of both
M. dirhodum and S. avenae. With this bioassay, we
investigated the preference of these two aphid species for
four winter wheat varieties with susceptibility to Fusarium
head blight (FHB) ranging between susceptible, mild and
tolerant. In a study of the LCG Belgium, a Flemish
research center for grains, different wheat varieties were
tested for their FHB susceptibility (Haesaert et al. 2009),
indicating that Lexus was a susceptible variety and Sahara
a tolerant variety. Homeros and Tulsa were moderately
susceptible and moderately tolerant, respectively. Different
parameters of the aphid preference behavior were scored.
The varieties were compared at seedling level, and for S.
avenae we also investigated the preference with regard to
ears as this aphid species is described to be ear attracted.
Differences in preference behavior are discussed in relation
to potential mechanisms for host selection and FHB susceptibility
to be attractive for both aphid species.
Materials and methods
Test organisms
Non-clonal cultures of S. avenae and M. dirhodum were
maintained on wheat seedlings. They were encouraged to
reproduce parthenogenetically under long-day conditions
(LD 18:6) at a constant temperature of 22°C.
Four varieties of winter wheat (Triticum aestivum L.)
were used: Sahara (---), Tulsa (--?), Homeros (-??)
and Lexus (???) where the (-) and (?) notations represent
their susceptibility for FHB ranging from tolerant
(---) to susceptible (???).
Experimental design for preference tests
To determine aphid preferences for wheat varieties, an
experimental design was developed. This was achieved
using a three-way connection linking two falcon tubes to
each other (Fig. 1a). Through the third opening of the
three-way connection, ten apterous adult aphids were
placed in the system using a small paint brush. All tests
were performed under long-day conditions (LD 18:6) at a
constant temperature of 22°C.
When the preference tests were conducted using 7-day-old
seedlings, 15-ml falcon tubes were used. Seeds were sterilized
in 1% NaOCl for 1 min, washed in sterile water and put into
4 g of sterile white sand with 0.5 ml of sterile water as shown
a
b
c
falcon tube
place where aphids are inserted
three-way connection
Fig. 1 Setup of the binary choice experiment: a schematic view,
b setup with seedlings, and c setup with ears
123

Preference of cereal aphids for winter wheat varieties 347
in Fig. 1b. The tubes were sealed with parafilm and placed in a
growth chamber (22°C, 16/8 light/dark cycle).
To perform preference tests at the ear level, the top of a
50-ml falcon tube was cut off in order to fit on the threeway
connection. As depicted in Fig. 1c, the ear was put
through the screw cap opening of the tube which was
afterward sealed with parafilm.
Preference of cereal aphids for different wheat varieties
at the seedling level
Binary choice experiments were conducted with seedlings of
the wheat varieties Sahara, Tulsa, Homeros and Lexus. After
the aphids were put in the preference meter, their choice was
scored every hour up to 7 h by counting the aphids on both
seedlings (‘first variety’, ‘second variety’) and in the threeway
connection (‘no choice made yet’). This was repeated
after 24 h. Each combination was tested five times. The
scores were analyzed using multinomial logistic regression
in SPSS version 15.0 (Statistical Package for Social Sciences).
This is an expansion of (binary) logistic regression
when the outcome of the categorical dependent had more
than two levels (Chan 2005). In this experiment, the
dependent factor ‘choice’ had three levels: ‘first variety’,
‘second variety’ and ‘no choice made yet’. A reference level
(for example ‘first variety’) was chosen and the other groups
(‘second variety’ and ‘no choice made yet’) were compared
with this reference. This was followed by taking another
reference (for example ‘second variety’) and compared again
(with ‘first variety’ and ‘no choice made yet’). This was
repeated until all levels were compared with each other.
Significant differences were identified at P = 0.05.
A second parameter was the speed of choice. For each
combination, a graph was drawn with x-axis = time and
y-axis = mean numbers of aphids that had made a choice.
A curve with a quadratic equation fitted these data points
best. The slope of this curve gave an indication of how
quickly the aphids made their choice.
Preference of S. avenae for different wheat varieties
at the ear level
The combinations of Homeros–Homeros, Lexus–Lexus
and Homeros-Lexus were investigated at the ear level with
S. avenae aphids as shown in Fig. 1c. The ears had not yet
reached anthesis GS 59 (Zadoks et al. 1974) and the
combinations were again tested five times. Every hour, the
level ‘no choice made yet’ was scored. Once all the aphids
had made a choice, they were counted by destroying the
ears. The same analysis of preference and speed of choice
was used as given above.
Results
Preference of cereal aphids for different wheat varieties
at the seedling level
For both aphid species, significant differences in preference
were found between the different winter wheat varieties
Fig. 2 Response in binary
choice experiment at seedling
level with different winter
wheat varieties (Lexus,
Homeros, Tulsa, Sahara) for M.
dirhodum. a Choice behavior at
seedling level over time.
Different letters above the bars
indicate a significant difference
in preference between the wheat
varieties. b Table with P values
over all variety combinations;
(-) and (?) indicate tolerance
and susceptibility for FHB,
respectively. c Average slopes
(±SE) with speed of choice for
all varieties over time
a
# aphids
c
slopes
10
9
8
7
6
5
4
3
2
1
0
Sahara
no choice made yet
Lexus
c
a
b
b
a
a
b
b
a
P=0.001
a
1h 4h 7h 24h
time
b
b
Sahara
(---)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6 7
time (h)
Tulsa
(- - +)
Homeros
(- + +)
Lexus
(+ + +)
Sahara
(- - -) 0.397 0.777 0.777 0.001
Tulsa
(- - +) 0.777 0.397 0.260
Homeros
(- + +) 0.777 0.209
Lexus
(+ + +) 0.777
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348 N. De Zutter et al.
(Figs. 2b and 3b). After 24 h, M. dirhodum preferred Lexus
over Sahara (P = 0.001) (Fig. 2a) and S. avenae showed
significant preference for Tulsa over Lexus (P = 0.014)
(Fig. 3a). S. avenae also preferred Tulsa over Homeros, but
this observation was not conclusive (P = 0.062).
When choosing between varieties at the seedling level,
the average slope ± SE after 1 h was 1.37 ± 0.23 for M.
dirhodum (Fig. 2c) and 0.76 ± 0.14 for S. avenae
(Fig. 3c).
Preference of S. avenae for different wheat varieties
at the ear level
Preference tests at the ear level were conducted where the
varieties of Homeros and Lexus were combined with each
other and among themselves. As depicted in Fig. 4a, no
significant preferences were found.
The slope after 1 h over all combinations at the ear level
for S. avenae was 3.45 ± 0.25 (Fig. 4b).
Discussion
To conduct preference tests, we developed a simple robust
preference meter. This was constructed out of falcon tubes
and a three-way connection. Using this tool, tests were set
up for cereal aphids S. avenae and M. dirhodum, which had
to make a choice between two seedlings or ears of wheat
varieties Sahara, Tulsa, Homeros or Lexus. If both aphid
species could choose between the same wheat varieties,
Fig. 3 Response in binary
choice experiment at seedling
level with different winter
wheat varieties (Lexus,
Homeros, Tulsa, Sahara) for S.
avenae. a Choice behavior at
seedling level over time.
Different letters above the bars
indicate a significant difference
in preference between the wheat
varieties. b Table with P values
over all variety combinations;
(-) and (?) indicate tolerance
and susceptibility for FHB,
respectively. c Average slopes
(±SE) with speed of choice for
all varieties over time
a
# aphids
c
slopes
10
9
8
7
6
5
4
3
2
1
0
b
Tulsa
no choice made yet
Lexus
P=0.014
a
a
a
a
b
ab
b
a
b
1h 4h 7h 24h
time
b
b
Sahara
(- - -)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6 7
time (h)
Tulsa
(- - +)
Homeros
(- + +)
Lexus
(+ + +)
Sahara
(- - -) 0.777 0.397 0.397 0.397
Tulsa
(- - +) 0.777 0.062 0.014
Homeros
(- + +) 0.777 0.260
Lexus
(+ + +) 0.777
a
Homeros
(- + +)
Lexus
(+ + +)
Homeros
(- + +) 0.572 0.777
Lexus
(+ + +) 0.777
b
slopes
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0 1 2 3 4 5
time (h)
Fig. 4 Response in binary choice experiment at ear level with
different winter wheat varieties (Lexus and Homeros) for S. avenae.
a Table with P values over all varieties combinations; (-) and (?)
indicate tolerance and susceptibility for FHB, respectively. b Average
slopes (±SE) with speed of choice for all varieties over time
123

Preference of cereal aphids for winter wheat varieties 349
there was no significant difference in choice found
(P [ 0.05), which was reflected by an equal amount of
aphids present in both tubes (Figs. 2b, 3b and 4a). This
illustrates the proof of concept. The distribution indicated
an unbiased design, meaning that group behavior could be
ruled out. The choice of one aphid was not affected by the
choice of another, because the remaining nine aphids did
not all choose the variety that the first aphid had chosen.
Significant preferences were found for both aphid species.
Apterous M. dirhodum preferred the variety Lexus
over Sahara and S. avenae preferred Tulsa over Lexus.
These results indicated that preference was an intrinsic
characteristic of cereal aphids, since both species did not
prefer the same winter wheat variety. When seedlings of
Homeros and Lexus were combined, no significant difference
in preference of S. avenae was found. The same
combination at the ear level also showed no significant
difference in preference.
According to the slopes of the quadratic equation for
determining how quickly the aphids made their choice, M.
dirhodum made their choice at the seedling level 1.8 times
faster than S. avenae. Once again, this indicates intrinsic
differences between the two aphid species. Although S.
avenae aphids decided more slowly at the seedling level,
they were able to make a choice 4.5 times faster at the ear
level than the seedling level, indicating a preference for
ears over seedlings. Indeed, Wratten (1975) reported that
aphids of S. avenae move rapidly to the ears when they
emerge. However, more research is needed to make firm
conclusions and to indicate the mechanisms behind this
behavior.
The preference tests as presented in this project can
contribute to a more reasoned choice of wheat varieties in
order to lower aphid infestations. However, it should be
noted that this study did not investigate the nutritional
influence of the different varieties on the aphids, or the
influence on their reproduction. It only broadens our
knowledge about aphid preference.
There are several mechanisms that play a role in the
choice behavior of aphids. Many studies demonstrate that
olfactory cues play a role in host finding by aphids. The
response of the two cereal aphids used in this project, S.
avenae and M. dirhodum, for plant volatiles was examined
in the study of Visser and Fu-shun (1995). With an electroantennogram,
it was found that (E)-2-hexenal and hexanal
elicit the largest responses in alate forms of both aphids.
However, while the corresponding alcohols elicited smaller
responses for S. avenae, the contrast between the responses
to aldehydes and alcohols were not so clear for M. dirhodum.
Another layer of complexity in the olfactometric
mechanism is that once aphids have settled on a host plant,
the volatile blend of that plant can be altered, changing the
decision of other aphids that have not yet settled. Quiroz
et al. (1997) pointed out that volatiles of wheat seedlings
infected with apterae of R. padi had a repelling effect and
several compounds were identified from seedlings with a
high aphid density, but not from the wheat seedlings alone.
Another host selection mechanism is that aphids can
visually discriminate between host and non-host targets.
This was confirmed by Gish and Inbar (2006) for apterous
Macrosiphoniella artemisiae aphids, which found their
way back to the host plant after dropping from the plant.
It is also possible that the aphid’s plant choice changes
when detecting a certain chemical after initial probing.
Nicol et al. (1992) pointed to a significant negative relationship
between the proportion of alate S. avenae colonizing
seedlings of different wheat varieties and the
hydroxamic acid levels in those varieties.
A remarkable observation in this project is that M. dirhodum
prefers the most FHB-susceptible variety (Lexus) over
the most FHB-tolerant one (Sahara). In contrast, S. avenae
prefers Tulsa, a moderately tolerant variety, over the susceptible
Lexus. Considering the experimental design we
used in this project, we suggest that all mechanisms playing
a role in host selection by aphids are taken into account.
This means that if there were different wheat characteristics
resulting from a different Fusarium susceptibility, they
have been taken into account in our preference meter.
This project elucidates the distribution of fungal pathogens
by aphids when they prefer an FHB-susceptible wheat
variety. When more aphids are located on a plant, variety or
plant part that is susceptible to a pathogen, there can be a
greater risk for the distribution of that pathogen. For
example, since M. dirhodum prefers the leaves of wheat,
this aphid species is more likely to play a role in the distribution
of leaf-living fungi such as Septoria sp. In contrast,
the ear-feeding S. avenae can also be a distributor of earattacking
fungi such as Fusarium sp. Although no clear link
was observed between susceptibility of wheat to FHB and
preference of aphids in the present study, it would be an
oversimplification to state at this point that there is no
preferential link between aphids and FHB-infected plants.
To date, knowledge on plant alternations of susceptible
varieties due to Fusarium infection is scarce, but likely to be
different from resistant varieties. In addition, evidence is
accumulating that mycotoxins produced by members of the
FHB disease complex can hijack the plant’s defense system
(Desmond et al. 2008; Gardiner et al. 2010), and in this way
influence the plant characteristics during the infection. A
structural insight into tritrophic interactions is warranted to
acquire a comprehensive view of the array of defenses that
wheat can use against fungal and insect invaders. In this
context, indirect evidence from the existence of tritrophic
interactions arises from another model system. Shore fly
adults (Scatella stagnalis (Fallen)) can contribute to the
aerial dispersal of three soil-borne plant pathogens
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350 N. De Zutter et al.
(Verticillium dahliae Kleb., Fusarium oxysporum
Schlechtend.:Fr. f. sp. basilici (Dzidzariya) Armstr. and
Armstr. and Thielaviopsis basicola (Berk. and Broome)
Ferraris). Consonant with this observation, they are attracted
to sporulating cultures of these pathogens and infected
plant tissues (El-Hamalawi 2008). The latter example suggests
that not only plant volatiles, but also fungal volatiles,
might be involved in the preference of insects.
In conclusion, the results of this project demonstrated
that there are intrinsic differences between aphids, in terms
of preference and speed of choice over different wheat
varieties and aphid species, even when they occur on the
same host plant. It was also demonstrated that the preference
of M. dirhodum and S. avenae for wheat varieties
differs according to the FHB susceptibility.
Acknowledgments The authors acknowledge support by the Special
Research Council of Ghent University and would also like to
thank Annelies De Roissart for her help in designing the preference
meter. N.D.Z. is holder of a Ph.D. grant by the Agency for Innovation
by Science and Technology in Flanders (IWT).
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