Amer J of Potato Res (2005) 82:197-201
197
Soybean Aphid, Aphis glycines Matsumura, a N e w Vector of P o t a t o Virus Y in P o t a t o J e f f r e y A. Davis*, E d w a r d B. R a d c l i f f e , a n d D a v i d W. R a g s d a l e Department of Entomology, University of Minnesota, 219 Hodson Hail, 1980 Folwell Ave., St. Paul, MN 55108, USA *Corresponding author: Tel: (612) 624-2751; Fax: (612) 625-5299; Email:
[email protected]
ABSTRACT S o y b e a n aphid (Aphis glycines Matsumura),
INTRODUCTION an
Since its discovery in the North Central region of the
e x o t i c s p e c i e s first d i s c o v e r e d in t h e N o r t h Central
United States in 2000, soybean aphid (Aphis glycines Mat-
region o f t h e U n i t e d S t a t e s in 2000, is a c o m p e t e n t vec-
sumura) has spread to 21 states (Venette and Ragsdale 2004).
tor o f s e v e r a l Potyviridae. S o y b e a n aphid has high
This aphid can directly damage soybean (Glycine m a x L.), cans-
f e c u n d i t y and p r o d u c e s alatae ( w i n g e d m o r p h s ) readily,
ing early leaf senescence and reducing pod set, seed quality, and
characteristics typical o f p r o f i c i e n t virus vectors. W h e n
yield (Xibei et al. 1994; Wang et al. 1996, 1998; Wu et al. 1999).
s o y b e a n aphids w e r e e x p o s e d t o Potato virus Y (PVY)-
In addition to direct feeding damage to soybean, soybean
i n f e c t e d p o t a t o p l a n t s and t h e n clip-caged o n h e a l t h y
aphid transmits several plant viruses including Alfalfa mosaic
p o t a t o p l a n t s in groups o f five or as single aphids, PVY
.virus (AMV), Cucumber mosaic virus (CMV), Peanut mottle
t r a n s m i s s i o n ranged f r o m 14% to 75% across all experi-
virus (PMV), Soybean mosaic virus (SMV), Tobacco ringspot
m e n t s . PVY°, PVYN, and PVYNTNstrains w e r e t r a n s m i t t e d
v i r u s (TRSV), and Tobacco vein-banding mosaic virus (TBMV) (Fang et al. 1985; Alleman et al. 2002; Clark and Perry
by s o y b e a n aphid.
2002; Wang and Ghabrial 2002). Soybean aphid is a proficient
RESUMEN
vector of nonpersistent viruses in part because of its high fecundity, short generation time (McCornack et al. 2004), and
E1 ~Lfido de la s o y a (Aphid glycines Matsumura),
propensity to form alatae (winged morphs) (Voegtlin and
e s p e c i e e x 6 t i c a que f u e r a d e s c u b i e r t a e n la regi6n nor-
Onstad 2003). Soybean aphid is congeneric with two known
central de los E s t a d o s U n i d o s e n el afio 2000 es un vec-
Potato virus Y (PVY) vectors: buckthorn aphid (Aphis nas-
tor de varios Potyviridae. E1 ~fido de la s o y a t i e n e una
turtii Kaltenbach) and cotton aphid (Aphis gossypii Glover).
alta f e c u n d i d a d y produce r ~ p i d a m e n t e f o r m a s aladas,
Aphis glycines and A. gossypii have been reported to produce
caracterlstica tipica de los v e c t o r e s de virus. C u a n d o los
hybrids which, while viable, have reduced fecundity (Zhang
~fidos de la s o y a se p u s i e r o n e n c o n t a c t o con el virus Y
and Zhong 1982). For these reasons, we hypothesized that soy-
(PVY) e n plantas de papa i n f e c t a d a s y l u e g o se inser-
bean aphid could be a vector of PVY.
taron en p l a n t a s sanas en grupos de cinco o c o m o ~fidos
PVY exists as several strains that have been placed into
solitarios, la trasmisi(in de PVY fue de 14% a 75% en
groups based on their symptomology in potato (Solanum
t o d o s los e x p e r i m e n t o s . Las v a r i a n t e s PVY °, PVYN y
tuberosum L.) and tobacco (Nicotiana tabaccum L.) (de Bokx
PVYNTNf u e r o n t r a s m i t i d a s por el ~fido de la soya.
and Huttinga 1981; Kerlan et al. 1999). The common strain, PVY°, produces mosaic and leaf drop in potato and tobacco. A strain designated as tobacco veinal necrotic strain, PVYN, produces mottling and necrosis in potato (Chachulska et al. 1997; Kerlan et al. 1999) and severe necrosis in tobacco (de Bokx
Accepted for publication 12 October 2004. ADDITIONAL KEY WORDS: Solanum tuberosum, virus transmission efficiency
and Huttinga 1981). In potato, PVYN tends to cause a milder mosaic than does PVY° and in some cultivars is essentially asymptomatic (de Bokx and Huttinga 1981). A variant of PVYN
198
AMERICAN JOURNAL OF POTATO RESEARCH
can produce tuber necrosis in potato and has been designated as PVYNTN.This variant causes typical mosaic symptoms on
Vol. 82
A p h i d Colonies Aphid colonies maintained in the laboratory were used in
potato leaves, sunken necrotic rings on tubers, and leaf necrosis
all transmission experiments. The soybean aphid colony was
in tobacco (Le Romancer et al. 1994). Tubers may be infected
founded from aphids collected from soybean at UMore Park in
with PVYNv~ but not show symptoms due to cultivar differ-
2001 and reared on soybean (cv M96-133151) in an environmen-
ences or lack of permissive environmental conditions. In addi-
tal growth chamber held at 25 ± 2 C, 75 ± 3% RH, and a pho-
tion, there are specific strains detected serologically as PVY°
toperiod of 16:8 (L:D). Colonies of green peach aphid and potato
that cause necrosis in tobacco (McDonald and Singh 1996;
aphid were founded from aphids collected from potato at
Chrzanowska 2001). These strains have been designated PVYN:°
UMore Park in 1996 and 2002, respectively, and reared on virus-
and are recombinants between PVY° and PVYN(Nie and Singh
free potato (various cultivars have been used) in a greenhouse
2002, 2003; Singh et al. 2003).
held at 20 ± 2 C, 50 ± 5% RH and a photoperiod of 16:8 (L:D). The
Preliminary experiments using a mass inoculation proce-
corn leaf aphid (Rhopalosiphum maidis (Fitch)) colony was
dure similar to the arena tests described by Irwin and Ruesink
founded from aphids collected from wheat (Triticum aestivum
(1986) suggested that soybean aphid can acquire and transmit
L.) at the St. Paul campus of the University of Minnesota in 2003
PVY (Davis et al. 2004). Objectives of this study were (i) to
and reared on barley (Hordeum vuLgare L.) in a greenhouse
confirm that soybean aphid can transmit PVY from potato to
held at 20 ± 2 C, 50 ± 5% RH and a photoperiod of 16:8 (L:D).
potato and (ii) to measure transmission efficiency.
Virus D e t e c t i o n
MATERIALS AND METHODS
Virus detection was performed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using
Virus S t r a i n s a n d M a i n t e n a n c e
polyclonal and monoclonal kits according to the manufac-
The PVY inoculum source used in these experiments orig-
turer's protocols (Agdia, Elkhart, IN, USA). Absorbance was
inated from PVY-infected tubers (cvs Red La Soda and Chief-
measured at 405 nm with a BioRad 450 Plate Reader (Her-
tain) harvested at the University of Minnesota Outreach,
cules, CA, USA). Plants were considered PVY positive if the
Research and Education Park (UMore Park), Rosemount, MN,
OD405 reading was three times negative controls (noninfected
from field plots used to screen potato breeding lines for PVY
plants). Viral RNA was extracted using a Qiagen (Valencia, CA,
resistance. In the field, this PVY source is maintained by annual
USA) RNeasy Plant Mini Kit. RT-PCR was performed with a
harvesting of tubers from the "infector rows," which are
Qiagen OneStep RT-PCR Kit using a competitive multiplex
blended with certified seed and planted as inoculum the next
approach with five primers based on nucleotide differences in
year. Thus, this source is representative of aphid-transmitted
the P1 gene of PVY to discriminate among strains (Nie and
PVY strains found in Minnesota, a characteristic considered
Singh 2002) and a six primer competitive multiplex to discrim-
desirable in transmission efficiency trials (Irwin and Ruesink
inate recombinant PVYN:° (Nie and Singh 2003). Primers spe-
1986; Power and Gray 1995; Gray et al. 1998; Lucio-Zavaleta et
cific to the coat protein and nuclear inclusion protein b region
al. 2001). All three PVY strains and recombinant PVYN:° were
created by Moravec et al. (2003) were also used to confirm
shown by reverse transcriptase polymerase chain reaction
strain type. This allowed simultaneous identification of all PVY
(RT-PCR) to be present in the inoculum source, but titer levels
strains present in plants having multiple strain infections.
for each strain were not determined. In the greenhouse, trans-
Bioassays to confirm PVYN detection involved rubbing leaves
missions to maintain the PVY source were by green peach
of tobacco, cv Samsun NN, with carborundum (silicon car-
aphid (Myz~v persicae (Sulzer)), and potato aphid (Macrosi-
bide, 600 grit) and plant sap or with water (control).
phum euphorbiaz (Thomas)). Aphids were allowed to move freely from infected to healthy potatoes, transmitting PVY. This ensured that freshly infected plants were available to
P o t a t o Test P l a n t s Potato plants were grown from minitubers, cv Katadhin
serve as virus sources for all experiments and that all PVY
(provided courtesy of J. P. Helgeson, Univ. Wisconsin, Madi-
strains maintained were aphid transmitted.
son), or true potato seed, cv GS-201 (from National Research Support Project-6, United States Potato Genebank, Sturgeon
DAVIS et al.: N E W V E C T O R O F POTATO VIRUS Y
2005
199
Bay, WI, USA), planted in 10-cm-diameter plastic pots contain-
ciency, single aphid experiments included three k n o w n PVY
ing sterile potting mix (Sun Gro Horticulture, Elma, Manitoba,
vectors, green p e a c h aphid, potato aphid, and corn leaf aphid.
Canada) and 5 g O s m o c o t e (14-14-14), a slow-release fertilizer
Transmission efficiency was estimated as n u m b e r of
(The Scotts Company, Marysville, OH, USA). Plants were held
infected test plants divided by total n u m b e r of test plants,
in a greenhouse at 20 _+ 2 C, 50 + 5% RH and a photoperiod of
expressed as a percentage. For groups of five aphids, the max-
16:8 (L:D). Prior to use in transmission experiments, test
i m u m likelihood estimator (MLE) was also used (Gibbs and
plants were serologically tested by DAS-ELISA to confirm that
G o w e r 1960; Venette et al. 2002). MLE was calculated as 1-(1-
they w e r e PVY-free.
x/m) ~/k,where x was the n u m b e r of test plants positive for PVY, m was the total n u m b e r of test plants, and k was the n u m b e r
Transmission Efficiency Tests
of aphids used per test plant. Differences in infection rates b e t w e e n test plants and
Adult apterae (wingless aphids) w e r e used in all transmission tests. Transmission efficiencies w e r e measured using
controls w e r e calculated by PROC NPARIWAY using the
aphids in groups of five or individually and virus-free potato
Wilcoxon score (SAS Institute, Cary, NC, USA). Wilcoxon
test plants grown from either minitubers (greenhouse-grown
score is a signed rank test, substituting for the paired t-test, in
prenuclear seed potatoes) or true potato seed. Soybean aphids
which treatments are ranked (positive or negative in this case)
were given a 4-h pre-acquisition fast after which they were
and c o m p a r e d (Snedecor and Cochran 1989). Tiffs test is used
placed on detached PVY-infected potato leaves in closed petri
w h e n assumptions for normality are not met, i.e., variance is
dishes and allowed an acquisition access period of 10 min. All
not constant (Snedecor and Cochran 1989).
excised leaves used as inoculum sources w e r e later tested by RT-PCR to identify PVY strains present. Aphids w e r e then
RESULTS
placed into 1.2-mm-diameter circular clip cages on PVY-free test plants. Test plants and aphids w e r e held in 0.2 m 3 "no-see-
In four experiments w h e r e test plants w e r e exposed to
um"-screened (Venture Textiles, MA, USA) cages, two plants
groups of five aphids, PVY was detected in 50% of the test plants
per cage, and aphids w e r e given an tmlimited inoculation
(range 37.5% to 75%) (Table 1). After exposure to a single aphid
access period. Surviving aphids were r e m o v e d manually after
in five experiments, PVY was detected in 33.3% of test plants
5 days. Plants w e r e retested for PVY by RT-PCR 2 w k after
(range 14.3°/5 to 50%) (Table 1). PVY infection was detected in
aphids w e r e removed. To provide comparisons of vector effi-
one of the control plants (1/16) grown from cv Katahdin mini-
TABLE 1--Soybean aphid transmission of PVY : ...
Experiment
:" r--
Test cultivax~
7=-~.----
PVY strains in inoculum
:::
:::~-=::"-"7
PVY transmission to test plants Control Source (not PVY(PVY inoculated) inoculated)
Strain frequency in test plants where transmission occurred ~ PVY° PVYN P ~ PVYN:°
Five aphid experiments 1 2 3 4
Katahdin Katahdin Katahdin Katahdin
O, N, NTN, N:O O, N, NTN, N:O O, N, NTN, N:O O, N, NTN, N:O
0/4 0/4 0/4 1/4 1/16(
6/8 4/8 3/8 3/8 16/32¢
6 4 2 2 14
6 4 1 2 13
0 4 1 2 7
0 0 0 0 0
O, N, NTN, N:O O O O O, N, NTN, N:O
0/8 0/3 0/8 0/7 0/5 0/31~
3/8 1/7 1/4 2/4 5/13 12/36¢
3 1 1 2 4 11
1
0
0
0 l
1 1
0 0
Single aphid exper~ments 1 2 3 4 5
GS 201 GS 201 GS 201 GS 201 GS 201
~Multiple PVY strains were often detected in infected plants so cumulative strain numbers can exceed total infected plants. bGrown from minitubers and true potato seed. cPVY transmission between control and inoculum sources in five aphid experiments and in single aphid experiments differed significantly, Wilcoxon Signed Rank Test, P = 0.03, and P = 0.013, respectively.
200
AMERICAN JOURNAL OF POTATO RESEARCH
Vol. 82
tubers, but in none of the plants (0/31) grown from cv GS201
the PVY° and PVYTM strains in this study were similar to that
true seed. Differences in infection incidence between test
reported by other authors (Katis and Gibson 1985; Harrington et
plants and controls were significantly different in both the five
al. 1986; Fereres et al. 1993), all of whom found that green peach
aphid and single aphid experiments; P -- 0.03, and P = 0.013,
aphid transmitted PVY° more efficiently than PVYN. If PVYN were to become abundant in the North Central
respectively. Efficiency of PVY transmission by soybean aphid ranged
region, this could force potato seed certification programs to rely
from 14% to 75% across all experiments. Calculated MLE for
to a much greater extent on serological testing because PVYN
groups of five aphids was 13.8%. In single aphid experiments,
infections tend to be more difficult to recognize visually. Soybean
PVY transmission by green peach aphid was 100°/0(8/8) and by
aphid appeared to transmit PVYN:°in preliminarymass inoculation
potato aphid 87.5% (7/8). There was no transmission of PVY by
experiments (Davis et al. 2004), but this recombinant was not
corn leaf aphid (0/8). The results for vectors other than A.
transmitted in experiments with groups of five or single aphids.
glycines were similar to efficiencies reported for these species in the literature (Ragsdale et al. 2001).
Soybean is not a host of PVY and even when soybean aphid had access to PVY inoculmu, its efficiency of transmis-
Soybean aphid transmitted strains PVY°, PVYN, and
sion was less than that of green peach aphid and potato aphid.
PVYNTNto potato (Table 1). Among the test plants positive for
However, potatoes are frequently rotated with soybean in the
PVY in the groups of five aphids and single aphid experiments,
North Central region, so this crop is often grown in close prox-
89°/0 (25/28) test plants were positive for PVY°, 58% (14/24)
imity to potatoes. Bird cherry-oat aphid (Rhopalosiphum padi
were positive for PVYN, and 33% (8/24) were positive for PVYm~.
(L.)), like soybean aphid, is an inefficient PVY vector (11.5%)
This indicates that soybean aphid can transmit either single or
compared to M. persicae, but has been implicated as a key vec-
multiple strains of PVY. There was no detectable transmission
tor of that virus in our region (DiFonzo et al. 1997). What soy-
of p\~N:~) in any experiment.
bean aphid lacks in transmission efficiency, it makes up in
Bioassays indicated that PVY~ was present in the inoculum.
sheer numbers. In 2003, 2.8 million ha of soybeans in Minnesota
Typical tuber symptoms of PVYm~, raised rings or arches that
were hffested with soybean aphid, with nearly 1.4 million ha
become cracks or sunken pits, were observed in the inoculum
treated to prevent yield losses. Voegtlin and Onstad (2003) esti-
tubers, but in the transmission experiments, test plants were
mated that -400 million soybean aphid alatae emigrated from
not grown to tuber bulking.
one 32-ha Illinois soybean field in a single day. Given this aphid's propensity to form alatae, its high fecundity, and rela-
DISCUSSION
tive transmission efficiency, soybean aphid appears to have the potential to be an important vector of PVY. This is the first
The role soybean aphid may play in PVY epidemiology in
confirmation of soybean aphid transmitting PVY.
the North Central region is uncertain, but there is cause for concern. In this study, soybean aphid had PVY-transmission
ACKNOWLEDGMENTS
efficiencies similar to other Aphis spp. considered important vectors of PVY. For example, Aphis nasturtii transmits PVY at
The authors acknowledge Dr. Roger Moon for his statistical
19°/0 to 50% and A. gossypii at 12% to 31% (Ragsdale et al.
advice. We also thank Dr. Philip Berger, USDA-APHIS-PPQ-
2001). Green peach aphid is considered to be the most efficient
CPHST, Raleigh, NC, Dr. Jeffrey Wyman, University of Wiscon-
PVY vector, whereas potato aphid is intermediate (Harrington
sin, Madison, WI, and anonymous peer reviewers for their
et al. 1986; Piton 1986; Harrington and Gibson 1989) and corn
constructive comments on a previous draft of this manuscript.
leaf aphid is inefficient (Halbert et al. 2003). Placed on this continuum, soybean aphid would fall between potato aphid
LITERATURE CITED
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