Knowing the enemy - the genetic diversity and host range of Turnip
yellows virus (TuYV), an important pathogen of oilseed rape in Europe
Max Newbert Supervisors: Dr. John Walsh, Dr. Graham Teakle, Dr. Guy Barker
School of Life Sciences, University of Warwick, Wellesbourne, Warwick, CV35 9EF
(m.newbert@warwick.ac.uk)
4. TuYV Incidence (continued)
1. Project aims
Investigate the genetic diversity of Turnip yellows virus (TuYV)
• This information will be used to plot virus movement and divergence across Europe.
• Obtaining the whole genome sequences of TuYV will be important for the synthesis of an infectious clone.
• Measuring genetic diversity of TuYV will be important for evaluating plant resistances.
Study host range
• The epidemiology of TuYV is not well studied and consequently the host range (crop plants and weeds) of TuYV
is being investigated.
• Host adaptation of different genotypes will be investigated.
2. Introduction
TuYV is a Polerovirus within the family Luteoviridae. As an RNA virus with a high mutation rate, TuYV evolves
rapidly. It causes major losses in vegetable brassicas (16-65% reduction in yield) and oilseed rape (OSR) (up to
30%) in the UK. Infection affects all components of OSR yield, including number of pods per plant, number of
seeds per pod and the oil content per seed (Hardwick et al., 1994).
C3
65
S188
Cw728
54
K4
65
50
69
52
8
S26
Brassica yellows virus BJS
3
81
B
ay
rassic
A JS
virus
ellows
62
99
12
U41
42
1
F9 8
99
AB
47
81
4
J
97
s
ow
l
l
ye
us
vir
2
ica
Cw
24
W -C4 1
s
as
Br
94
61
s
J
BB
C2
s
B ra
a
sic
lo w
yel
s
v ir u
P1
A full-length infectious clone of TuYV will
be constructed in order to investigate
genome diversity and its effect on
virulence and host range.
W-S
2
0
P9
B
C2 1
74
G5
A
0
59
49
Insecticide treatments can be effective if applied before aphid flights, but do not offer a complete solution, as one
feed from one aphid can spread TuYV. Plant resistance to TuYV would reduce costs and provide better protection.
62
F58
TuYV is transmitted by aphids. Myzus persicae (Fig. 3) is probably the main vector in the UK; it feeds on a variety
of crops, including brassicas. Once aphids have acquired the virus, they carry it for the rest of their lives.
LA
B
P6
52
Figure 2A. TuYV-infected oilseed rape leaves showing no symptoms. B. Transmission electron micrograph of TuYV spherical particles (~25nm
diameter) (Colin Clay, University of Warwick). C. Muted symptoms are typical of abiotic stress, and are not diagnostic (Stevens et al., 2008).
Knock-outs of genes in Arabidopsis that
might be involved in essential TuYV
functions are being investigated in order
to identify novel sources of resistance.
Any resistances identified will be
evaluated using TuYV isolates from the
different genetic groups. Any plant
genes identified could then be targeted
in crops.
V
99
TuYV is a genetically diverse virus.
Sequencing ORF0 indicated three major
phylogenetic groups. ORF5 sequences
have shown greater diversity (Table 3)
and suggest a different group structure
to that based on ORF0.
Tu
Y
W-
C
hi
d
7
B
or
c
L1
93
A
ia
n
99
Figure 1. Structure of TuYV genome (adapted from Veidt et al., 1988). Open Reading Frames (ORF) functions: ORF0, host range, suppressor of gene
silencing; ORF1/2, RNA dependent RNA polymerase; ORF3, major coat protein; ORF4, transport protein; ORF5, minor coat protein, virus accumulation
and persistence within the vector.
L1
ORF3
V -F
ORF1
3’
st
ra
l
99
ORF5
ORF4
T uY
5’
ORF2
Phylogenetic analyses were performed
(e.g. on ORF5, Fig. 5). Comparing distinct
genetic groups with biological properties
could provide information on
functionality of TuYV genes, for example,
indicating host determinates. TuYV has
been isolated from a number of weeds
and sequenced, indicating greater
diversity than detected in crop plants.
Au
09
ORF0
5. Genetic diversity
G2
TuYV does not always cause obvious symptoms (Fig. 2A), possibly due to it being limited to the phloem of plants.
Symptoms that can occur, include yellowing, or purpling of the leaves and stunting of plants. TuYV has a +sense
RNA genome of 5.6kb with six open reading frames (Fig. 1), with ORF 3 and ORF 5 encoding the coat proteins,
producing small ~25nm spherical particles (Fig. 2B). Infection often goes unrecognised as environmental stresses
can cause similar symptoms (Fig. 2C).
Figure 4. Sites sampled around Europe. Sample sites were selected in major OSR-growing regions.
Figure 5. ORF5 nucleotide phylogenetic tree shows 4 genetic groups of European TuYV
(Black/Red/Blue/Purple). Isolate origin codes: C = Cheshire, Cw = Cornwall, K = Kent, L =
Lincolnshire, S = Suffolk, F = France, G = Germany, P = Poland, U= Ukraine, W- =Weed
Table 3. Amino acid identity between the TuYV-FL and European isolates
compared to the amino acid identity shared between European Isolates.
TuYV
Gene
Amino acid % identity
of European isolates
with TuYV-FL
Amino acid % identity
between European isolates
excluding TuYV-FL
P0
89
96
P1
89
93
P2
91
96
P3
96
97
P4
96
98
P5
56
92
Figure 3. Myzus persicae. A. Scanning electron micrograph of the aphid (Colin Clay, University of Warwick). B. Brassica plant colonised by the aphids.
4. TuYV Incidence in OSR in Europe
6. Host Range
Sites across Europe have been sampled (Fig. 4). Leaf samples from plants were tested by ELISA to determine TuYV
infection. From the five UK sites sampled so far, infection was up to 76% in 2012 and 92% in 2013 (Table 1). A
reservoir TuYV population has been detected in weeds, threatening vegetable and oilseed brassicas.
TuYV has a wide host range; crop and
weed species are being investigated
in the UK (Table 4).
TuYV incidences in the UK were lower in spring 2013 than spring 2012 (Table 1). Reduced numbers of M. persicae
were caught in Rothamsted Insect Survey suction traps in autumn 2012, relative to autumn 2011; this may have
been due to the wet and cool autumn of 2012. TuYV incidences in other parts of Europe were high in both years
(Table 2); up to 99% infection was detected. Such high levels of infection would have a dramatic effect on yield,
highlighting the need for effective control measures.
TuYV isolated from weeds is being
assessed by aphid transmission for
the ability to infect OSR, in order to
identify reservoirs and host range.
Table 1. UK incidence of TuYV In weeds and OSR in 2012 and 2013.
Map
location
Fig. 4
Sampling site
1
Cheshire,
Gawsworth
76
5
2012
Common Chickweed
1
Cheshire,
Knutsford
30
0
2012
-
1
Cheshire,
Northwich
46
5
2012
Shepherd's Purse
Suffolk,
Lavenham
68
Suffolk,
Cockfield
66
2
Suffolk, Acton
70
0
3
Kent,
Chartham
70
3
Kent,
Whitfield
22
2
2
TuYV
incidence
in OSR (%)
TuYV
Year
incidence in
weeds (%)
Weed species
infected
Table 2. Incidence of TuYV in OSR in other parts of Europe in
2012 and 2013.
Map
Sampling site
Year
TuYV
location
incidence
Fig. 4
(%)
Understanding host range is
important for defining plant-virus
interactions and understanding the
epidemiology of the virus.
Table 4. Weeds found to be infected with TuYV and ability of these isolates to infect OSR.
TuYV isolate
Host origin
Ability to infect OSR*
W-S51
W-S52
W-S53
W-S62
W-S121
W-S127
W-C261
W-C412
W-K547
W-K548
W-K553
W-K559
W-K560
W-K616
W-K626
W-Cw683
W-Cw684
W-Cw694
W-Cw695
W-Wa1035
W-Wa1039
Shepherds Purse
Groundsel
Shepherds Purse
Weld
Shepherds Purse
Garlic Mustard
Chickweed
Shepherds Purse
Ribwort Plantain
Purple Archangel
Ribwort Plantain
Ribwort Plantain
Winter speedwell
Mayflower
Cleavers
Field Pansy
Winter Speedwell
Shepherds Purse
Shepherds Purse
Common Pepper Weed
Thistle
Yes
Yes
Yes
No
Yes
No
Yes
Yes
No
No
Yes
Yes
No
No
No
No
Yes
No
Yes
No
No
6
Aires (France)
2012
78
7
Chartres (France)
2012
92
8
Courcelles (France)
2012
73
9
Bulquoy (Belgium)
2012
66
10
Marburg (Germany)
2012
(98)*
Shepherd's Purse,
Common Groundsel,
Weld
11
Peine (Germany)
2012
(96)
12
Sulbeck (Germany)
2012
(100)
Garlic Mustard,
Shepherd's Purse
13
Bergtheim (Germany)
2012
(90)
14
Dyngby (Denmark)
2012
(10)
2012
-
15
Abildgard (Denmark)
2012
(0)
0
2013
-
16
Holeby (Denmark)
2012
(77)
25
2013
Plantain, Purple
Archangel, Winter
Speedwell
17
Lviv (Ukraine)
2012
(30)
6. Conclusions/Future work
18
Kiev (Ukraine)
2012
(0)
19
Liverdy en Brie (France)
2013
93
• TuYV is an important and widespread virus that can cause significant reductions in the yields of vegetable
and oilseed brassicas.
20
10
2012
2012
3
Kent, Folkston
92
10
2013
May Flower, Cleavers
20
Einbeck (Germany)
2013
97
4
Warwickshire,
Gaydon
38
0
2013
-
21
Kondratowice (Poland)
2013
99
4
Warwickshire,
Gaydon
20
0
2013
-
4
Warwickshire,
Gaydon
46
10
2013
Common Pepper
Weed, Thistle
5
Cornwall,
Tredethy
0
20
2013
Wild Pansy, Winter
Speedwell, Shepherd's
Purse
5
Cornwall,
Lansallos
12
0
2013
-
5
Cornwall,
Tregony
14
0
2013
-
* Bracketed % figures are not true incidences, as tested
leaves were not definitely from different plants – bulk
samples received.
References:
-Hardwick, N. V., Davies, J. M. and Wright, D. M. (1994). Plant Pathology 43, 1045-1049
-Veidt, I., Lot, H., Leiser, M., Scheidecker, D., Guilley, H., Richards, K. and Jonard, G. (1988). Nucleic Acids Research 16, 9917-993.
-Stevens, M., McGrann, G., & Clark, B., (2008). In: HGCA Research Review No. 69.
-Rothamsted insect survey bulletin. Accessed March 2012: http://www.rothamsted.ac.uk/insect-survey/STAphidBulletinArchive.php?Year=2013
This will be expanded to include a
study of other crop plants to
determine whether they are
susceptible to TuYV isolates from
OSR.
*Myzus persicae were fed on leaves of weed plants that had tested positive for TuYV in
ELISA and then transferred to OSR. Three weeks later, the OSR plants were tested for
TuYV by ELISA.
• Due to aphid numbers, aphid resistance to insecticides and the nature of TuYV transmission, insecticide
treatments do not offer complete protection against the virus and hence alternative control measures
such as natural plant resistance are needed.
• It is important to have a measure of the genetic diversity of TuYV in order to rigorously evaluate the
sources of resistance to TuYV we have identified in earlier work.
• Whole genome analysis will also be useful for measuring diversity in order to test resistances already
identified in brassicas and any identified in Arabidopsis knock-out studies, for spectrum of resistance.
• Whole genome sequences will assist in the construction of full-length infectious viral clones.
Acknowledgements:
•Supervisors: Dr. John Walsh, Dr. Graham Teakle, Dr. Guy Barker
•Dr. Adam Baker, Dr. Elvis Asare-Bediako, Charlotte Nellist, Isabel Saunders
Collaborators
•Dr. Peter Werner (KWS), Dr. Jo Bowman (Nickersons), Mark Nightingale (Elsoms), Dr. Richard Jennaway
(Saaten Union), Sean Burns (Syngenta), Matthew Clarke (Monsanto), Prof. Rod Snowdon (Justus Liebig
University), Keith Norman (Velcourt)
•Funding from BBSRC and the Perry Foundation