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PLANT RESISTANCE
Characterization of Eight Russian Wheat Aphid (Hemiptera:
Aphididae) Biotypes Using Two-Category Resistant–Susceptible
Plant Responses
G. J. PUTERKA,
1,2
S. J. NICHOLSON,
1
M. J. BROWN,
1
W. R. COOPER,
3
F. B. PEAIRS,
4
AND T. L. RANDOLPH
4
J. Econ. Entomol. 107(3): 1274Ð1283 (2014); DOI: http://dx.doi.org/10.1603/EC13408
ABSTRACT Eight biotypes of the Russian wheat aphid, Diuraphis noxia (Kurdjumov), have been
discovered in the United States since 2003. Biotypes are identiÞed by the distinct feeding damage
responses they produce on wheat carrying different Russian wheat aphid resistance genes, namely,
from Dn1 to Dn9. Each Russian wheat aphid biotype has been named using plant damage criteria and
virulence categories that have varied between studies. The study was initiated to compare the plant
damage caused by all the eight known Russian wheat aphid biotypes, and analyze the results to
determine how Russian wheat aphid virulence should be classiÞed. Each Russian wheat aphid biotype
was evaluated on 16 resistant or susceptible cereal genotypes. Plant damage criteria included leaf roll,
leaf chlorosis, and plant height. The distribution of chlorosis ratings followed a bimodal pattern
indicating two categories of plant responses, resistant or susceptible. Correlations were signiÞcant
between chlorosis ratings and leaf roll (r
2
⫽0.72) and between chlorosis ratings and plant height (r
2
⫽
0.48). The response of 16 cereal genotypes to feeding by eight Russian wheat aphid biotypes found
RWA1, RWA2, RWA6, and RWA8 to differ in virulence, while Russian wheat aphid biotypes RWA3,
RWA4, RWA5, and RWA7 produced similar virulence proÞles. These biotypes have accordingly been
consolidated to what is hereafter referred to as RWA3/7. Our results indicated that the Þve main
biotypes RWA1, RWA2, RWA3/7, RWA6, and RWA8 can be identiÞed using only four wheat
genotypes containing Dn3,Dn4,Dn6, and Dn9.
KEY WORDS plant resistance, wheat, barley, invasive specie, plant damage
Biotypes of the Russian wheat aphid, Diuraphis noxia
(Kurdjumov), have become a serious threat to the
development and deployment of plant resistance in
cereals since the occurrence of the Þrst Russian wheat
aphid biotype (RWA2) in 2003 (Haley et al. 2004).
Previous to the appearance of RWA2, resistance genes
for wheat designated Dn1 to Dn9, Dnx, and Dny were
found to be effective against the original Russian
wheat aphid (RWA1) that invaded the United States
in 1986 (Haley et al. 2004). Six additional biotypes
(RWA3ÐRWA8) have been described (Burd et al.
2006, Weiland et al. 2008, Randolph et al. 2009) since
the discovery of RWA2. The biotypic diversity in Rus-
sian wheat aphid has limited useful resistant germ-
plasm to 94M370 (Dn7 gene), CI2401, and STARS
2414-11. In addition, two sources of resistance in bar-
ley, STARS 9301B and STARS 9577B, still exhibit
strong resistance to all eight biotypes (Puterka et al.
2006, Mornhinweg 2012).
The appearance of RWA2 was unexpected because
the original invading population of Russian wheat
aphid (RWA1) was considered to be asexual (Butts
1992, Hammon and Peairs 1998, Burd et al. 1998) and
showed no biotypic (Puterka et al. 1992) or genetic
variation (Puterka et al. 1993, Shufran et al. 1997). In
2005, an area-wide study on Russian wheat aphid bio-
typic variation in wheat found RWA2 to have almost
completely displaced the original biotype, RWA1
(Puterka et al. 2007). Genetic variation among Russian
wheat aphid biotypes suggested a single (Shufran et al.
2007, Shufran and Payton 2009) or multiple invasions
into the United States that afterward diversiÞed into
more biotypes (Liu et al. 2010). Sexually reproducing
Russian wheat aphid have also been discovered in the
high plateau region of western Colorado, in which
⬎40 distinct biotypes were documented when 90
progeny were screened against key sources of Russian
wheat aphid resistance (Puterka et al. 2012). The re-
cent discovery of the currently designated biotypes,
Mention of trade names or commercial products in this article is
solely for the purpose of providing speciÞc information and does not
imply recommendation or endorsement by the United States Depart-
ment of Agriculture. The authors declare no conßicts of interest.
1
USDAÐARS, Plant Science Research Laboratory, 1301 N. West-
ern, Stillwater, OK 74074.
2
Corresponding author, e-mail: gary.puterka@ars.usda.gov.
3
USDAÐARS, Fruit & Vegetable Insect Research, 5230 Konnowac
Pass Road, Wapato WA 98951.
4
Department of Bioagricultural Sciences and Pest Management,
1177 Campus Delivery, Colorado State University, Fort Collins, CO
80523-1177.
the potential for new biotype introductions, and the
production of new biotypes via sexual reproduction
present a signiÞcant challenge for breeding Russian
wheat aphid-resistant wheat with durable resistance
to a range of biotypes.
Past studies that characterized and designated the
eight Russian wheat aphid biotypes compared new
biotypes with a limited subset of already known bio-
types or with those biotypes available at the time.
Biotypes RWA3, 4, and 5 were characterized by the
mean damage ratings (1 [no damage] to 9 [dead plant
rating]) they caused on plant leaves (Burd et al. 2006),
while Russian wheat aphid biotypes RWA6, 7, and 8
were characterized by a combination of leaf damage
and leaf roll ratings (Weiland et al. 2008, Randolph et
al. 2009). Although these biotype studies categorized
plants into resistant, intermediately resistant, or sus-
ceptible damage categories, the damage rating range
for each category differed. In some cases, damage
ratings produced by Russian wheat aphid biotypes on
certain plant genotypes were statistically similar but
were assigned different damage categories. Further
complicating biotype classiÞcations were studies that
reported opposing damage responses in identical
wheat genotypes; for example, RWA3 feeding damage
on Dn7 (Burd et al. 2006, Weiland et al. 2008, Randolph
et al. 2009).
Producing reproducible biotype effects on cereal
germplasm is fundamental to the identiÞcation of Rus-
sian wheat aphid biotypes and the detection of new
more virulent biotypes. The objective of this study was
to characterize the damage caused by all the eight
Russian wheat aphid biotypes (RWA1ÐRWA8) on re-
sistant and susceptible wheat and barley genotypes.
Damage was assessed using rating scales for leaf chlo-
rosis and leaf roll, similar to those used in previous
biotype studies (Burd et al. 2006, Weiland et al. 2008,
Randolph et al. 2009). The damage rating distributions
were analyzed to determine whether a two- (resistant
and susceptible) or three (resistant, intermediately
resistant, and susceptible)-category system was most
appropriate for classifying Russian wheat aphid viru-
lence to each plant genotype. Data on plant damage
components were analyzed with the goal of develop-
ing virulence categories for biotypes that account for
the variability in damage data. Consistencies and dis-
crepancies among previously reported virulence pro-
Þles to common wheat genotypes were addressed in
light of our results to better unify the biotype concept
for Russian wheat aphid and facilitate studies on bio-
typic diversity.
Materials and Methods
The original Russian wheat aphid biotype (RWA1)
that invaded the United States was collected in Bailey
County, TX, in 1986 (Burd et al. 1993) and maintained
by the U.S. Department of AgricultureÐAgricultural
Research Service (USDAÐARS), Stillwater, OK. Bio-
types RWA3 (Floyd County, TX), RWA4 (Lubbock
County, TX), and RWA5 (Park County, WY) were
originally collected in 2002Ð2003 and described by
Burd et al. (2006). Four biotypes were obtained from
the Colorado State University, Ft. Collins, CO, namely,
RWA2 collected in Baca County, CO, in 2003 (Haley
et al. 2004) and RWA6, RWA7, and RWA8 collected
in western (RWA6 and RWA8) and eastern (RWA7)
Colorado (Weiland et al. 2008). These biotypes were
obtained by U.S. Department of AgricultureÐAgri-
cultural Research ServiceÐPlant Science Research
Laboratory (USDAÐARSÐPSRL) at Stillwater, OK,
soon after they were collected. For the 7 yr before
this study, the eight Russian wheat aphid biotypes
were maintained on ÔYumaÕ wheat grown in 8-cm-
diameter pots within cylindrical clear plastic cages
5 cm in diameter and 30 cm in length that were
topped with a Þne mesh cloth for ventilation. The
aphid colonies were held in a room with tempera-
tures of 20Ð22⬚C on light racks with a photoperiod
of 14:10 (L:D) h provided by four 40W cool white
ßuorescent lights.
The virulence and biotypic classiÞcations for isofe-
male colonies of Russian wheat aphid biotypes 1Ð8
were determined by plant reactions of 16 Russian
wheat aphid-resistant or -susceptible wheat and bar-
ley genotypes (Table 1). Seeds of each genotype were
planted in a mini-ßat 18 cm in width by 27 cm in length
by 5 cm in depth in a completely randomized design.
The mini-ßats were placed in a 27 cm in width by 38
cm in length by 6 cm in depth tray Þlled with con-
struction grade sand that served as a bed to seat metal
frame cages (30 cm in width by 40 cm in length by 30
cm in height) covered with Þne mesh nylon screen.
The plants were watered moderately and infested at a
rate averaging 10 aphids per plant genotype when the
plants reached a height of 3Ð5 cm. The experiment was
replicated 10 times during the fall and spring of 2009Ð
2010 under variable greenhouse temperatures (12Ð
28⬚C) and sunlight (10Ð13 h). Plant damage was as-
sessed as a percent leaf chlorosis or necrosis on a 1Ð9
rating scale of increasing damage (1 ⫽healthy; 2 ⫽
1Ð5% and spotted; 3 ⫽5Ð20%; 4 ⫽21Ð35%; 5 ⫽36Ð50%;
6⫽51Ð65%; 7 ⫽66Ð80%; 8 ⫽81Ð95%; and 9 ⫽
96 Ð100% or dead (Webster et al.1991, Burd et al. 2006)
when susceptible ÔYumaÕ and ÔCusterÕ plant genotypes
rated an 8Ð9 (20Ð24 d). Leaf rolling was rated using a
1Ð3 scale, where 1 ⫽ßat; 2 ⫽folded or partially rolled;
and 3 ⫽fully rolled (Burd et al. 1993). The distribution
and frequency of leaf chlorosis ratings for all 16 ge-
notypes across all aphid biotypes was tested against a
normal distribution (SAS Institute 2010) to determine
if the distribution was modally or normally distributed
across the nine damage scores to classify aphid viru-
lence. Each plant genotypeÐRussian wheat aphid bio-
type combination was classiÞed as either resistant
(R ⫽damage rating of ⱕ5) or susceptible (S ⫽damage
rating of ⬎5) based on the bimodal distribution of the
chlorosis rating data. Categorical data from leaf chlo-
rosis, plant infestation level, and rolling for aphid bio-
types within and across cereal genotypes were sub-
jected to a one-way nonparametric analysis using a
KruskalÐWallis analysis of variance (ANOVA; PROC
NPAR1WAY) and, if signiÞcant (P⬎
2
ⱕ0.05), plant
comparisons were made using pair-wise KruskalÐWal-
June 2014 PUTERKA ET AL.: EIGHT RUSSIAN WHEAT APHID BIOTYPES CHARACTERIZATION 1275
lis tests (SAS Institute 2010). Parametric data from
plant heights were analyzed by a two-way ANOVA to
determine the effects of aphid biotype, plant geno-
type, and their interaction; means for biotypes within
genotypes were compared using the least signiÞcant
difference (LSD) analyses (Pⱖ0.05).
Results
The frequency distribution of all chlorosis ratings
for 16 plant genotypes and eight Russian wheat aphid
biotypes (n⫽1280) indicated a bimodal distribution
in the data (Fig. 1). Therefore, the nonparametric
nature of the chlorosis rating data was best repre-
sented by a two-categoryÑresistant or susceptibleÑ
plant response to Russian wheat aphid feeding. Dam-
age ratings for the resistant plant category (n⫽590)
ranged from 1 to 5 with an average of 2.97 ⫾0.04.
Resistant ratings primarily encompassed 2Ð4, which
represented 1Ð35% leaf chlorosis. The susceptible
damage category (n⫽690) encompassed damage
scores ranging from 4 to 9 with a mean of 7.67 ⫾0.05.
This distribution was mainly represented by damage
scores ranging from 6 to 9 (51⫺100% chlorosis) with
a damage rating of 9 representing 39% of the total
scores in the susceptible damage category. Compari-
son of distributions showed the susceptible damage
category slightly overlapped with the resistant damage
category for ratings 4 (n⫽2) and 5 (n⫽27). For this
reason, the chlorosis rating of 5 provided a reasonable
dividing point between cereal resistance (rating 1 to
ⱕ5) and susceptibility (⬎5Ð9). If an intermediate cat-
egory was added, the damage ratings would have had
a distribution frequency for ratings 4 ⫽138, 5 ⫽61, and
Table 1. List of resistant and susceptible wheat and barley genotypes used to determine Russian wheat aphid virulence
Crop Plant
genotype
Resistance source
or gene
Resistance to
Russian wheat
aphid biotype
Reference
Wheat CO03797 Dn1 1 Haley et al. (2004)
CO03804 Dn2 1 Haley et al. (2004)
CO03811 Dn3 1 Haley et al. (2004)
Yumar Dn4 1 Haley et al. (2004), Collins et al. (2005)
CO950043 Dn5 1 Haley et al. (2004)
CO960223 Dn6 1 Haley et al. (2004)
94M370 Dn7 1Ð8 Haley et al. (2004), Weiland et al. (2008), Randolph et al. (2009)
Karee-Dn8Dn8 1 Haley et al. (2004)
Betta-Dn9Dn9 1 Haley et al. (2004)
CI 2401 PI372129 1Ð8 Collins et al. (2005), Weiland et al. (2008), Randolph et al. (2009)
STARS 2414-11 PI366515 1Ð8 Collins et al. 2005, Weiland et al. 2008, Randolph et al. 2009
Custer Ð Susc. Haley et al. (2004)
Yuma Ð Susc. Haley et al. (2004)
Barley STARS 9301B Rdn1/Rdn2/Rdn3 1Ð8 Mornhinweg (2012), Mittal et al. (2009)
STARS 9577B Rdn1/Rdn2 1Ð8 Mornhinweg (2012)
Mittal et al. (2008)
Schyler Ð Susc. Mornhinweg et al. (1999)
Fig. 1. Frequency distribution for all chlorosis damage ratings from 16 plant genotypes and eight biotypes (n⫽1280).
Mean damage ratings for each biotypeÐplant genotype combination were classiÞed as resistant or susceptible to determine
the distributions of the underlying damage rating data.
1276 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3
6⫽129, which is not the expected normal distribution
that would support it as a valid category.
Analysis of chlorosis ratings for each plant genotype
pooled across all biotypes showed highly signiÞcant
differences (Pⱕ0.0001) between resistant (R ⫽dam-
age rating 1Ð5) and susceptible (S ⫽damage rating
⬎5Ð9) categories for wheat resistance genes Dn1 to
Dn6 and Dn9 (Table 2). The remaining plant geno-
types produced uniform chlorosis damage responses
that were either resistant (e.g., STARS 2414-11) or
susceptible (e.g., Yuma). Comparison among plant
genotypes within each Russian wheat aphid biotype
showed signiÞcant differences in leaf chlorosis ratings
(P⬎
2
ⱕ0.0001;
2
⫽110.4Ð135.3, df ⫽15; Table 3).
The susceptible standards Yuma, Custer, and ÔSchylerÕ
were among the highest damaged of the cereal geno-
types within each biotype. The wheat genotypes
94M370 (Dn7), CI2401, and STARS 2414-11, and the
barley genotypes STARS 9301B and STARS 9577B,
were highly resistant to all eight Russian wheat aphid
biotypes, as indicated by low chlorosis ratings. Signif-
icant differences in chlorosis ratings were found
among Russian wheat aphid biotypes within most
cereal genotypes (range, P⬎
2
⫽0.006 to ⬍0.0001;
2
⫽19.2Ð45.8; df ⫽7) with the exception of suscep-
tible Custer and highly resistant STARS 2414-11 (P⬎
2
⫽0.055Ð0.70;
2
⫽4.6⫺13.2; df ⫽7, respectively).
RWA2 was the most virulent to RWA1-resistant cereal
genotypes followed by a group with lesser virulent
biotypes RWA3, 4, 5, and 7. Least virulent to the
RWA1-resistant cereal genotypes was RWA1 followed
by slightly more virulent RWA6 and RWA8.
The leaf roll ratings produced by Russian wheat
aphid biotypes signiÞcantly differed for cereal geno-
types containing Dn1 to Dn6 genes, Dn9, and STARS
9577B (P⬎
2
⬍0.0001;
2
⫽87.4 Ð137.1; df ⫽15; Table
4). No signiÞcant differences in leaf roll ratings were
found among Russian wheat aphid biotypes within
cereal genotypes that were highly susceptible (e.g.,
ÔYumarÕ) or highly resistant (e.g., 94M370 [Dn7]) in
Table 4. There was a moderate correlation between
increasing chlorosis damage ratings and an increase in
leaf roll damage ratings (P⬍0.0001; r
2
⫽0.72).
Plant height was signiÞcantly impacted by cereal
genotype (F⫽129.08; df ⫽15, 1438; P⬍0.0001),
Russian wheat aphid biotype (plus uninfested control;
F⫽450.96; df ⫽8, 1438; P⬍0.0001), and their inter-
action (F⫽125.93; df ⫽120, 1438; P⫽0.0001; Table
5). In general, feeding by the Russian wheat aphid
biotypes reduced plant height of cereal genotypes by
⬇50Ð75% in comparison to the uninfested controls.
There was a signiÞcant yet inconsistent inverse linear
relationship between chlorosis ratings and plant
height (F⫽1343.47; df ⫽1439; P⬍0.0001; r
2
⫽0.48).
Virulence patterns of each Russian wheat aphid
biotype were categorized into resistant (R) and sus-
ceptible (S) plant responses for the 16 cereal geno-
types (Table 6) based on chlorosis ratings in Table 3.
Wheat genotypes containing the Russian wheat aphid
resistance genes Dn1, Dn2, Dn3, Dn5, Dn7, and Dn8
responded similarly to all Russian wheat aphid bio-
types. In contrast, Dn4, Dn6, and Dn9 showed vari-
ability in plant responses to feeding by Russian wheat
aphid biotypes. The wheat germplasm sources, CI2401
and STARS 2414-11, were highly resistant to all Rus-
sian wheat aphid biotypes. The wheat (Custer and
Yuma) and barley (Schyler) varieties that were used
as positive controls conÞrmed uniform susceptibility
to all Russian wheat aphid biotypes. The barley lines
STARS 9301B and STARS 9577B displayed strong re-
sistance to the all Russian wheat aphid biotypes. Over-
all, variable damage responses in four wheat geno-
types containing Dn3, Dn4, Dn6, and Dn9 identiÞed
RWA1, RWA2, RWA6, and RWA8, respectively, as
unique biotypes, whereas RWA3, 4, 5, and 7 did not
differ in their responses (Table 7).
Table 2. Chi-square analysis of chlorosis damage ratings (nⴝ80) produced by all Russian wheat aphid biotypes within each cereal
genotype after categorizing each rating as resistant (R ⴝ1to<5) or susceptible (>5–9)
Cereal
genotype Resistance gene
a
Mean ⫾SE damage rating per category
(observations per category)
2
P⬎
2
(df ⫽1)
Resistant Susceptible
Wheat
CO03797 Dn1 3.65 ⫾0.31 (20) 6.18 ⫾0.17 (60) 19.1 ⬍0.0001
CO03804 Dn2 4.05 ⫾0.20 (20) 7.11 ⫾0.21 (60) 20.7 ⬍0.0001
CO03811 Dn3 3.80 ⫾0.13 (20) 7.48 ⫾0.19 (30) 41.7 ⬍0.0001
Yumar Dn4 4.06 ⫾0.15 (30) 6.80 ⫾0.21 (50) 40.5 ⬍0.0001
CO9500043 Dn5 4.06 ⫾0.24 (20) 7.00 ⫾0.18 (60) 35.0 ⬍0.0001
CO960223 Dn6 3.18 ⫾0.14 (70) 6.50 ⫾0.22 (10) 23.8 ⬍0.0001
94M370 Dn7 2.85 ⫾0.10 (80) Ð Ð Ð
Karee-Dn8 Dn8 Ð 7.16 ⫾0.17 (80) Ð Ð
Betta-Dn9 Dn9 3.50 ⫾0.16 (10) 6.11 ⫾0.19 (70) 15.8 ⬍0.0001
CI2401 New 3.10 ⫾0.14 (80) Ð Ð ⬍0.0001
STARS 2414-11 New 2.31 ⫾0.01 (80) Ð Ð Ð
Yuma Sus. Ð 8.85 ⫾0.05 (80) Ð Ð
Custer Sus. Ð 8.85 ⫾0.04 (80) Ð Ð
Barley
STARS 9301B Rdn1/Rdn2/Rdn3 2.37 ⫾0.06 (80) Ð Ð Ð
STARS 9577B Rdn1/Rdn2 2.61 ⫾0.08 (80) Ð Ð Ð
Schyler Sus. 8.48 ⫾0.09 (80) Ð Ð
a
Sus., susceptible to all Russian wheat aphid biotypes; new, new resistant germplasm.
June 2014 PUTERKA ET AL.: EIGHT RUSSIAN WHEAT APHID BIOTYPES CHARACTERIZATION 1277
Table 3. Mean leaf chlorosis damage ratings taken 20 –24 d after cereal genotypes were infested by Russian wheat aphid biotypes
Cereal
genotype Resistance gene
a
Biotype
b
RWA1 RWA2 RWA3 RWA4 RWA5 RWA6 RWA7 RWA8
Wheat
CO03797 Dn1 3.9 ⫾0.2deC 6.0 ⫾0.3fB 5.9 ⫾0.2dB 5.8 ⫾0.3eB 6.2 ⫾0.3cdAB 6.3 ⫾0.3cAB 6.9 ⫾0.4cdA 3.4 ⫾0.2efgBC
CO03804 Dn2 4.2 ⫾0.3deC 7.0 ⫾0.4deB 8.2 ⫾0.4abA 7.8 ⫾0.4cA 7.0 ⫾0.4bcB 6.9 ⫾0.5cB 7.1 ⫾0.5cdAB 3.9 ⫾0.2cdeC
CO03811 Dn3 3.5 ⫾0.2efgC 7.9 ⫾0.3bcdA 8.0 ⫾0.4bA 8.0 ⫾0.3bcA 6.9 ⫾0.3bcB 7.7 ⫾0.4bAB 7.8 ⫾0.4bcA 4.1 ⫾0.2cdC
Yumar Dn4 4.0 ⫾0.3deC 7.3 ⫾0.5cdeAB 7.6 ⫾0.4bA 6.7 ⫾0.2dAB 7.0 ⫾0.2bcAB 3.8 ⫾0.2deC 6.5 ⫾0.5dB 4.2 ⫾0.2cC
CO9500043 Dn5 4.4 ⫾0.3dC 7.3 ⫾0.4cdeAB 7.9 ⫾0.4bA 7.0 ⫾0.2dAB 6.8 ⫾0.2bcB 7.0 ⫾0.2cAB 7.7 ⫾0.4bcAB 3.3 ⫾0.3efgD
CO960223 Dn6 3.6 ⫾0.2efB 6.8 ⫾0.2efA 3.9 ⫾0.2eB 3.1 ⫾0.2fB 3.1 ⫾0.4dB 3.9 ⫾0.3dB 3.6 ⫾0.2eB 3.5 ⫾0.3defB
94M370 Dn7 2.8 ⫾0.2gABC 2.5 ⫾0.2dBC 2.2 ⫾0.1gfC 2.4 ⫾0.2ghBC 3.2 ⫾0.4dA 3.4 ⫾0.3defA 3.0 ⫾0.2eAB 2.9 ⫾0.2fghAB
Karee-Dn8 Dn8 7.4 ⫾0.5bBC 7.1 ⫾0.4cdeCD 6.4 ⫾0.3cdD 6.6 ⫾0.3dCD 7.3 ⫾0.3bBC 8.0 ⫾0.2bAB 8.5 ⫾0.2abA 8.3 ⫾0.3bA
Betta-Dn9 Dn9 6.6 ⫾0.4cAB 6.6 ⫾0.2efAB 6.8 ⫾0.3cAB 6.4 ⫾0.3dBC 6.9 ⫾0.2bcAB 6.8 ⫾0.3cAB 7.3 ⫾0.4cdA 3.4 ⫾0.2efgC
CI2401 New 2.8 ⫾0.2gA 2.8 ⫾0.3gA 2.9 ⫾0.3fA 2.7 ⫾0.3fgA 2.9 ⫾0.3deA 3.4 ⫾0.2defA 3.3 ⫾0.2efA 3.1 ⫾0.2fgA
STARS 2414-11 New 2.2 ⫾0.2gA 2.2 ⫾0.2gA 2.0 ⫾0.2gA 2.2 ⫾0.1ghA 2.5 ⫾0.3deA 2.7 ⫾0.3fgA 2.4 ⫾0.2fA 2.3 ⫾0.2hA
Yuma Sus. 8.9 ⫾0.1aAB 8.9 ⫾0.1aAB 8.8 ⫾0.1aAB 8.5 ⫾0.2abB 8.6 ⫾0.2aAB 9.0 ⫾0.0aA 9.0 ⫾0.0aA 9.0 ⫾0.0aA
Custer Sus. 8.9 ⫾0.1aA 8.7 ⫾0.3abA 8.8 ⫾0.1aA 8.9 ⫾0.1aA 8.8 ⫾0.1aA 8.8 ⫾0.1aA 9.0 ⫾0.0aA 8.9 ⫾0.1abA
Barley
STARS 9301B Rdn1/Rdn2/Rdn3 2.9 ⫾0.1gfA 2.4 ⫾0.2gBC 2.0 ⫾0.0gB 2.0 ⫾0.0gB 2.2 ⫾0.1eAB 2.6 ⫾0.2gAB 2.5 ⫾0.2fAB 2.4 ⫾0.2hBC
STARS 9577B Rdn1/Rdn2 3.0 ⫾0.31fgAB 2.4 ⫾0.3gBC 2.1 ⫾0.2gC 2.4 ⫾0.2ghBC 2.7 ⫾0.2deABC 3.1 ⫾0.1efgA 2.4 ⫾0.2fBC 2.8 ⫾0.2ghAB
Schyler Sus. 8.8 ⫾0.1aA 8.0 ⫾0.3bcB 7.9 ⫾0.1bB 8.2 ⫾0.3bcAB 8.4 ⫾0.3aAB 8.9 ⫾0.1aA 8.9 ⫾0.1aA 8.8 ⫾0.1abA
Chlorosis ratings ranged from 1 ⫽no damage to 9 ⫽95Ð100% chlorotic of necrotic leaf.
a
Sus., Russian wheat aphid susceptible control; new, new RWA2 resistant germplasm.
b
Means within a column followed by the same lower-case letter, or within a row followed by the same upper-case letter, are not signiÞcantly different (P⬎0.05, KruskalÐWallis test).
1278 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3
Table 4. Comparisons of mean leaf roll damage ratings taken 20 –24d after cereal genotypes were infested by Russian wheat aphid biotypes
Cereal
genotype Resistance gene
a
Biotype
b
RWA1 RWA2 RWA3 RWA4 RWA5 RWA6 RWA7 RWA8
Wheat
CO03797 Dn1 1.1 ⫾0.1deB 2.3 ⫾0.6bcA 2.1 ⫾0.2bcA 2.1 ⫾0.2cA 2.0 ⫾0.0cA 2.1 ⫾0.4cA 2.1 ⫾0.4cA 1.7 ⫾0.4cA
CO03804 Dn2 1.6 ⫾0.4cdB 2.4 ⫾0.3bcA 2.6 ⫾0.4aA 2.4 ⫾0.2bcA 2.8 ⫾0.2abA 2.9 ⫾0.1aA 2.9 ⫾0.1aA 2.2 ⫾0.2abA
CO03811 Dn3 1.7 ⫾0.4bcCD 2.4 ⫾0.4bcAB 2.8 ⫾0.2aAB 2.8 ⫾0.2aAB 2.8 ⫾0.2abAB 2.2 ⫾0.2cBC 3.0 ⫾0.0aA 1.4 ⫾0.1aD
Yumar Dn4 1.6 ⫾0.2cdBC 2.3 ⫾0.4bcA 2.9 ⫾0.2aA 2.9 ⫾0.2aA 3.0 ⫾0.0aA 1.1 ⫾0.1abC 2.6 ⫾0.4abA 1.8 ⫾0.3abB
CO9500043 Dn5 1.5 ⫾0.2edC 2.4 ⫾0.4bcB 3.0 ⫾0.0aA 2.9 ⫾0.1aAB 2.6 ⫾0.2bAB 2.4 ⫾0.4bcB 2.7 ⫾0.3abAB 2.8 ⫾0.2aAB
CO960223 Dn6 1.2 ⫾0.2edB 2.6 ⫾0.3abA 1.5 ⫾0.3dAB 1.5 ⫾0.3dAB 1.0 ⫾0.0dB 1.3 ⫾0.3dAB 1.3 ⫾0.3dAB 1.2 ⫾0.2cdB
94M370 Dn7 1.0 ⫾0.0eA 1.0 ⫾0.6eA 1.0 ⫾0.0eA 1.0 ⫾0.0eA 1.0 ⫾0.0dA 1.0 ⫾0.0dA 1.0 ⫾0.0dA 1.1 ⫾0.1eA
Karee-Dn8 Dn8 2.7 ⫾0.4aAB 2.4 ⫾0.4aB 2.5 ⫾0.2abAB 2.6 ⫾0.2abAB 2.5 ⫾0.2bAB 2.9 ⫾0.1aAB 3.0 ⫾0.0aA 2.9 ⫾0.1aAB
Betta-Dn9 Dn9 2.2 ⫾0.3bB 2.9 ⫾0.1aA 2.3 ⫾0.3bcB 2.3 ⫾0.6bcB 2.6 ⫾0.2abB 2.4 ⫾0.2bcB 2.6 ⫾0.2abB 1.4 ⫾0.1aC
CI2401 New 1.0 ⫾0.0eA 1.0 ⫾0.0eA 1.0 ⫾0.0eA 1.2 ⫾0.2edA 1.0 ⫾0.0dA 1.2 ⫾0.2dA 1.0 ⫾0.0dA 1.4 ⫾0.4cdeA
STARS 2414-11 New 1.0 ⫾0.0eA 1.1 ⫾0.1eA 1.1 ⫾0.1deA 1.1 ⫾0.1edA 1.0 ⫾0.0dA 1.0 ⫾0.0dA 1.2 ⫾0.2dA 1.2 ⫾0.2deA
Yuma Sus. 3.0 ⫾0.0aA 3.0 ⫾0.2aA 2.8 ⫾0.2aA 2.9 ⫾0.1aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA
Custer Sus. 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA
Barley
STARS 9301B Rdn1/Rdn2/Rdn3 1.0 ⫾0.0eA 1.1 ⫾0.1eA 1.1 ⫾0.0deA 1.0 ⫾0.0eA 1.0 ⫾0.0dA 1.0 ⫾0.0dA 1.0 ⫾0.0dA 1.0 ⫾0.2eA
STARS 9577B Rdn1/Rdn2 1.1 ⫾0.1edB 1.6 ⫾0.6deA 1.1 ⫾0.1deB 1.0 ⫾0.0eB 1.0 ⫾0.0dB 1.0 ⫾0.0dB 1.0 ⫾0.0dB 1.0 ⫾0.2eB
Schyler Sus. 3.0 ⫾0.0aA 2.7 ⫾0.2abA 2.7 ⫾0.3aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA 3.0 ⫾0.0aA
Leaf roll was rated as 1 ⫽ßat leaf, 2 ⫽folded leaf, and 3 ⫽fully rolled leaf.
a
Sus., Russian wheat aphid susceptible control; new, new RWA2 resistant germplasm.
b
Means within a column followed by the same lower-case letter, or within a row followed by the same upper-case letter, are not signiÞcantly different (P⬎0.05, Kruskal Wallis test).
June 2014 PUTERKA ET AL.: EIGHT RUSSIAN WHEAT APHID BIOTYPES CHARACTERIZATION 1279
Table 5. Comparisons of mean plant heights of cereal genotypes taken 20 –24d after being infested with Russian wheat aphid biotypes in comparison to an uninfested control
Cereal
genotype Resistance gene
a
Biotype
b
RWA1 RWA2 RWA3 RWA4 RWA5 RWA6 RWA7 RWA8 Uninfested
Wheat
CO03797 Dn1 14.9 ⫾1.3B 15.1 ⫾0.9B 14.5 ⫾0.9B 13.7 ⫾0.9B 13.8 ⫾1.1B 14.4 ⫾0.7B 13.4 ⫾0.3B 14.8 ⫾1.0B 44.0 ⫾0.5A
CO03804 Dn2 17.3 ⫾1.7B 15.9 ⫾1.6B 17.0 ⫾2.0B 19.5 ⫾0.4B 20.6 ⫾1.2B 18.1 ⫾2.3B 16.6 ⫾2.0B 19.8 ⫾1.7B 43.6 ⫾7.6A
CO03811 Dn3 14.6 ⫾1.5C 13.7 ⫾1.3CD 14.5 ⫾0.9C 16.0 ⫾1.7BC 15.6 ⫾1.1BC 11.2 ⫾0.6DE 9.4 ⫾0.7E 18.0 ⫾1.6B 43.0 ⫾1.5A
Yumar Dn4 19.6 ⫾1.2B 18.5 ⫾1.3B 19.8 ⫾1.5B 20.7 ⫾1.2B 20.6 ⫾0.7B 18.3 ⫾1.6B 17.3 ⫾1.9B 19.1 ⫾2.8B 40.8 ⫾1.8A
CO9500043 Dn5 16.6 ⫾1.2B 12.9 ⫾0.7C 13.6 ⫾1.0BC 16.1 ⫾1.2BC 16.9 ⫾2.2B 14.9 ⫾0.6BC 14.4 ⫾1.3BC 16.0 ⫾0.6BC 40.6 ⫾1.6A
CO960223 Dn6 21.0 ⫾0.9CDE 16.8 ⫾1.1DE 15.7 ⫾1.2E 24.5 ⫾2.7BC 27.1 ⫾2.7B 19.0 ⫾1.5DE 16.5 ⫾1.4E 22.1 ⫾2.3BCD 59.3 ⫾1.9A
94M370 Dn7 29.7 ⫾2.5B 26.2 ⫾2.4B 29.5 ⫾2.3B 30.4 ⫾3.1B 28.4 ⫾4.7B 27.4 ⫾0.7bB 25.3 ⫾1.6B 27.6 ⫾1.7B 43.8 ⫾1.8A
Karee-Dn8 Dn8 12.4 ⫾1.4BC 12.9 ⫾1.5BC 14.3 ⫾0.5B 14.3 ⫾0.4B 12.8 ⫾0.2BC 11.8 ⫾0.7B 12.2 ⫾1.2BC 13.7 ⫾0.5BC 55.6 ⫾0.4A
Betta-Dn9 Dn9 12.5 ⫾1.3B 13.8 ⫾1.2B 14.1 ⫾0.6B 13.7 ⫾0.5B 13.5 ⫾1.2B 12.1 ⫾1.6B 11.7 ⫾1.2B 11.6 ⫾1.0B 52.6 ⫾1.7A
CI2401 New 19.3 ⫾1.5BC 17.7 ⫾1.5C 19.6 ⫾1.2BC 23.5 ⫾3.0B 22.0 ⫾0.7BC 19.1 ⫾1.9BC 17.1 ⫾1.6C 23.1 ⫾1.7B 46.6 ⫾2.9A
STARS 2414-11 New 29.5 ⫾2.1BC 27.9 ⫾1.0bBC 30.4 ⫾2.4BC 32.1 ⫾0.7B 33.0 ⫾2.8B 30.2 ⫾1.7BC 25.3 ⫾0.4C 27.3 ⫾0.4BC 38.8 ⫾1.4A
Yuma Sus. 15.2 ⫾1.3BC 14.6 ⫾0.8C 17.0 ⫾2.2BC 18.2 ⫾1.7B 17.5 ⫾1.8BC 15.9 ⫾1.7BC 15.5 ⫾2.4BC 17.5 ⫾1.4BC 42.8 ⫾0.2B
Custer Sus. 14.9 ⫾0.4B 15.0 ⫾0.4B 16.6 ⫾3.9B 14.2 ⫾1.1B 15.1 ⫾1.3B 15.6 ⫾6.1B 13.1 ⫾0.9B 14.5 ⫾0.8B 43.8 ⫾4.4A
Barley
STARS 9301B Rdn1/Rdn2/Rdn3 24.3 ⫾0.6C 26.7 ⫾1.5CD 36.1 ⫾1.9BC 38.8 ⫾1.8B 39.2 ⫾5.4B 34.4 ⫾2.4BCD 26.5 ⫾4.5CD 26.1 ⫾4.8CD 48.8 ⫾3.2A
STARS 9577B Rdn1/Rdn2 35.1 ⫾3.0BC 30.6 ⫾1.5BC 35.7 ⫾0.9BC 37.1 ⫾6.2B 34.3 ⫾1.7BC 32.1 ⫾1.8BC 27.6 ⫾2.7C 26.9 ⫾2.9C 51.8 ⫾2.8A
Schyler Sus. 14.7 ⫾1.1B 12.7 ⫾1.6B 14.9 ⫾0.5B 15.4 ⫾1.8B 14.3 ⫾1.2B 13.1 ⫾1.7B 14.0 ⫾0.2B 12.5 ⫾0.8B 44.0 ⫾1.2A
a
Sus., Russian wheat aphid susceptible control; new, new RWA2 resistant germplasm.
b
Means within a row followed by the same upper-case letter are not signiÞcantly different (Pⱖ0.05, LSD test).
1280 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 3
Discussion
The strong bimodal frequency distribution of leaf
chlorosis ratings for the 16 cereal genotypes reßected
a resistantÐsusceptible chlorosis rating relationship to
Russian wheat aphid virulence. Clustering of the chlo-
rosis ratings toward the lower and higher ends of the
1 to 9 rating scale and a small frequency of overlap
between the resistant and susceptible distributions
approaching the intermediate rating of 5 supported a
two-category plant damage response (Fig. 1). Non-
parametric analyses of the two-category data con-
Þrmed that the differential responses of cereal geno-
types to Russian wheat aphid biotypes were highly
signiÞcant (P⬎
2
ⱕ0.0001; Table 2). Those cereal
genotypes that responded differently to aphid feeding
(e.g., Dn1 to Dn6) had resistant or susceptible cate-
gories separated by a difference of ⬇2 damage ratings
(30% chlorosis or necrosis) and supported signiÞcant
differences between the biotypes abilities to damage
cereal genotypes (Table 3). Those plant genotypes
with uniform responses to the Russian wheat aphid
biotypes showed clear-cut high levels of either resis-
tance or susceptibility. Inclusion of an intermediate
category of 4Ð5.9 would have clearly resulted in chlo-
rosis ratings being assigned different plant response
categories even though they did not differ signiÞ-
cantly (e.g., Table 3; intermediate for RWA4 on Dn1 ⫽
5.8B; susceptible for RWA6 on Dn1 ⫽6.3AB). These
conßicts were also evident in other studies that used
an intermediate category to make comparisons among
Russian wheat aphid biotypes (Weiland et al. 2008,
Randolph et al. 2009).
Previous studies used either leaf chlorosis (Burd et
al. 2006) or leaf chlorosis plus leaf roll ratings
(Weiland et al. 2008, Randolph et al. 2009) to classify
aphid virulence to cereal genotypes. Leaf roll (Table
4) was moderately correlated (P⬍0.0001; r
2
⫽0.72)
with chlorosis ratings in both wheat and barley, thus,
was not a useful measure for Russian wheat aphid
virulence. This result is supported by other studies
that found no signiÞcant correlation between leaf roll-
ing and chlorosis ratings (Smith et al. 2004, Burd et al.
2006). Plant height (Table 5) was weakly correlated
(r
2
⫽0.48; P⬍0.0001) with chlorosis ratings. In con-
trast, plant stunting was reported to best describe the
quantitative damage response to RWA1 infestations in
cereals, although growth reductions occurred in both
resistant and susceptible germplasm. In general, plant
height was reduced by ⬇50% for resistant and ⬇75%
for susceptible responses for the cereal genotypes in
our study. These reductions support Þndings in similar
studies on barley (Puterka et al. 2006) and wheat
(Puterka et al. 2013) under greenhouse conditions.
However, the lack of a strong correlation between
plant height and leaf chlorosis made plant height an
unreliable factor for discriminating Russian wheat
aphid biotypes. Therefore, we used only leaf chlorosis
ratings to determine the virulence of Russian wheat
aphid biotypes to the cereal genotypes.
Table 6. Summary of resistant(R) and susceptible (S) responses of the plant genotypes based on chlorosis damage ratings produced
by Russian wheat aphid biotypes 20 –24d after infestation
Cereal
genotype
Biotype
Resistance gene
a
RWA1 RWA2 RWA3 RWA4 RWA5 RWA6 RWA7 RWA8
Wheat
CO03797 Dn1 RSSSSSSR
CO03804 Dn2 RSSSSSSR
CO03811 Dn3 RSSSSSSR
Yumar Dn4 RSSSSRSR
CO9500043 Dn5 RSSSSSSR
CO960223 Dn6 RS RRRRRR
94M370 Dn7 RRRRRRRR
Karee-Dn8 Dn8 SSSSSSSS
Betta-Dn9 Dn9 SSSSSSSR
CI2401 New RRRRRRRR
STARS 2414-11 New RRRRRRRR
Yuma Sus. SSSSSSSS
Custer Sus. SSSSSSSS
Barley
STARS 9301B Rdn1/Rdn2/Rdn3 RRRRRRRR
STARS 9577B Rdn1/Rdn2 RRRRRRRR
Schyler Sus. SSSSSSSS
Chlorosis ratings (Table 3) ranging 1 to ⱕ5⫽resistant (R); ⬎5Ð9 ⫽susceptible (S).
a
Sus., susceptible to all Russian wheat aphid biotypes; new, RWA2 new resistant germplasm.
Table 7. Primary wheat differential needed to identify biotypes
RWA1 to RWA8
RWA
biotype
a
Wheat genotype
b
CO03797
(Dn3)
Yumar
(Dn4)
CO960223
(Dn6)
Betta-Dn9
(Dn9)
RWA1 R R R S
RWA2 S S S S
RWA3/7 S S R S
RWA6 S R R S
RWA8 R R R R
Biotypes that are grouped (RWA3/7) produced similar responses
on the cereal genotypes.
a
RWA3/7 represents a consolidation of RWA3, RWA4, RWA5, and
RWA7.
b
Chlorosis ratings of 1 to ⱕ5⫽resistant (R); ⬎5Ð9 ⫽susceptible
(S).
June 2014 PUTERKA ET AL.: EIGHT RUSSIAN WHEAT APHID BIOTYPES CHARACTERIZATION 1281
Our study and previous Russian wheat aphid bio-
typing studies used similar 1Ð9 leaf chlorosis ratings,
which enabled a basis for comparing results. The chlo-
rosis ratings in our study (Table 3) were generally near
the ratings previously reported for speciÞc biotypeÐ
plant genotype interactions (Haley et al. 2004, Burd et
al. 2006, Weiland et al. 2008, Randolph et al. 2009).
Although it is not possible to discuss every discrepancy
between Russian wheat aphid biotyping studies, two
types of discrepancies for Russian wheat aphid viru-
lence to cereal genotypes will be highlighted. One
type of inconsistency is when an intermediate re-
sponse was used, e.g., Yumar rating 5.3 and 5.8 to
RWA4 and RWA5, respectively (Randolph et al.
2009). Our study found Yumar was susceptible to all
three biotypes (rating 7.6Ð6.7), which would be
aligned with Randolph et al. (2009) results as being
susceptible if a two-category response were used.
Variations in plant response between studies can only
be partially resolved by reducing the plant categories
to resistant or susceptible. A second example is where
opposite results on the resistance status of a cereal
genotype occurred among studies whether an inter-
mediate resistance category was used or not. For ex-
ample, RWA5 feeding on Yuma was originally re-
ported as resistant (chlorosis rating 3.9; Burd et al.
2006) but our results (rating 8.6; Table 3) and another
study (rating 7.4; Randolph et al. 2009) found Yuma to
be susceptible. Another example is 94M370 (Dn7),
when fed upon by RWA3 and RWA4, was originally
reported to be susceptible (6.5Ð6.9, Burd et al. 2006)
but was highly resistant in our study (chlorosis rating
2.2Ð2.4; Table 3) and Randolph et al. (2009) (both
rating 1.0). The large differences between these stud-
ies damage ratings for Yuma and 94M370 appeared to
be anomaly, which is difÞcult to explain. Yet, these
original ratings were unusual and, in cases like these,
are best veriÞed through independent laboratory test-
ing. The two-category plant response classiÞcation
should be less prone to misclassiÞcations by account-
ing for inherent variation in plant responses to similar
aphid clones owing to biotic or abiotic factors that
inßuence plant responses, or simply variation owing to
sampler error.
The response of 16 cereal genotypes to feeding by
eight Russian wheat aphid biotypes showed no signif-
icant differences among Russian wheat aphid biotypes
RWA3, 4, 5, and 7 (Tables 3 and 6) when using resis-
tant or susceptible plant responses. Accordingly, these
biotypes have been consolidated to what is hereafter
referred to as RWA3/7 (Table 7). Our results support
a previous study that screened progeny from a sexually
reproducing Russian wheat aphid population and
found RWA3, 4, and 7 had similar virulence proÞles
(Puterka et al. 2012) and concluded they could be a
single biotype that consisted of different genotypes
that vary slightly in virulence. In summary, results
indicated that there are mainly Þve biotypes RWA1,
RWA2, RWA3/7, RWA6, and RWA8 that can be iden-
tiÞed using four wheat genotypes containing Dn3,
Dn4, Dn6, and Dn9. These results are comparatively
consistent with most of the previously reported results
and represent a consensus between our results and
other studies (Burd et al. 2006, Weiland et al. 2008,
Randolph et al. 2009). The potential for new biotypes
to occur via sexual reproduction (Puterka et al. 2012),
and the possibility for new biotype introductions into
the United States (Liu et al. 2010) poses signiÞcant
challenges to the development of durable resistance to
a range of Russian wheat aphid biotypes. Screening
RWA populations for biotypic diversity can be facil-
itate by use of four Russian wheat aphid resistance
sources (Table 7) that would identify the current
Russian wheat aphid biotypes, and by adding other
important sources of resistance (e.g., 94M370 (Dn7)
and STARS 2414-11) to identify new biotypes. Mon-
itoring Russian wheat aphid populations for shifts in
biotype composition and detecting new Russian
wheat aphid biotypes will be a crucial aspect in the
development and deployment of durable sources of
Russian wheat aphid resistance in cereals.
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Received 18 September 2013; accepted 21 March 2014.
June 2014 PUTERKA ET AL.: EIGHT RUSSIAN WHEAT APHID BIOTYPES CHARACTERIZATION 1283