Download Epidemiology and control of fungally transmitted cereal viruses

MINISTRY OF AGRICULTURE, FISHERIES AND FOOD
Date project completed:
Research and Development
31/12/1998
Final Project Report
(Not to be used for LINK projects)

Section 1 : Identification sheet
1.
(a)
MAFF Project Code
CE0504
(b)
Project Title
Epidemiology and control of fungally transmitted viruses of cereals
and their vectors
(c)
MAFF Project Officer
Dr David Cooper
(d)
Name and address
of contractor
IACR-Rothamsted
Harpenden
Herts
Postcode
(e)
Contractor’s Project Officer
Dr M J Adams
(f)
Project start date
01/04/1994
(g)
Final year costs:
(h)
Total project costs / total staff input:
Project end date
31/12/1998
approved expenditure
£51,000
actual expenditure
£51,000
approved
Project endproject
date expenditure
£290,000
actual project expenditure
£281,636
*approved
Project end staff
date input
4.7
*actual staff input
4.7
12/01/1999
(i)
Date report sent to MAFF
(j)
Is there any Intellectual Property arising from this project ?
*staff years of direct science effort
Section 2 : Scientific objectives / Milestones
CSG 13 (1/97)
AL5 2JQ
1
NO
2.
Please list the scientific objectives as set out in CSG 7 (ROAME B). If necessary these can be
expressed in an abbreviated form. Indicate where amendments have been agreed with the MAFF
Project Officer, giving the date of amendment.
(1) Determine the host specificity and properties that control survival, spread and infection, of isolates of
Polymyxa, including their ability to vector viruses and the effects of competition from other soil microbes.
(2) Compare and characterise isolates of Polymyxa by molecular methods and develop diagnostic
procedures based on nucleic acid methods.
(3) Characterise, in terms of biological and molecular properties, variant isolates of BaYMV and develop
methods for their diagnosis using nucleic acid based methods.
(4) Develop methods, based on current and future immunology and nucleic acid assays, for diagnosing
barley and oat infecting viruses and their vector in soil.
(5) To determine the basis of resistance to mosaic viruses in Japanese cultivars in current use by UK plant
breeders (added for final year)
3.
List the primary milestones for the final year.
It is the responsibility of the contractor to check fully that ALL primary milestones have
been met and to provide a detailed explanation if this has not proved possible
Milestones
Number
CSG 13 (1/97)
Target
date
Title
Milestones met?
in full
2
on time
1
2
To monitor the presence of BaYMV-2 in samples of
susceptible barley cultivars from different areas of
England
To determine the basis of resistance to mosaic
viruses in Japanese cultivars in current use by UK
plant breeders
31/03/1999
NO
NO
31/03/1999
YES
YES
If any milestones have not been met in the final year,
an explanation should be included in Section 5.
Section 3 : Declaration
4.
I declare that the information I have given in this report is correct to the best of my knowledge and belief. I
understand that the information contained in this form may be held on a computer system.
Signature
Name
Date
Professor R T Plumb
Position in Organistation
Head of Crop and Disease Management Department
Section 4 : Executive summary
CSG 13 (1/97)
3
11/01/1999
The purpose of this project is to provide information on the basic biology of the mosaic viruses of cereals, especially
barley mild mosaic virus (BaMMV) and barley yellow mosaic virus (BaYMV) and their fungus vector, Polymyxa
graminis. This includes studies of vector biology, variability and control; the viruses and their variability; methods for
diagnosis of virus in soil and the nature of cultivar resistance.
Studies of vector isolates showed that these grew best on barley and most (but not all of them) also infected wheat. The
majority of isolates tested, including most of those from the UK, were able to transmit the barley mosaic viruses, but a
few isolates did not. Soils with widely differing cropping histories were tested for their populations of Polymyxa parasitic
on barley. By mixing soils that had low (or undetectable) Polymyxa populations with one containing a large inoculum of
P. graminis and BaMMV, tests were made to detect any suppressive effects. None of the soils tested showed any
evidence of ability to suppress Polymyxa infection and it seems unlikely that biological control will prove to be a useful
option. Isolates of Polymyxa were characterised by nucleotide sequencing of ribosomal DNA. Isolates of P. graminis could
be allocated to one of three subgroups (only two of which are represented by temperate isolates). These distinctions may be
related to differences in isolate origin and ability to transmit virus. These DNA sequences were used to design sets of
PCR primers for use in diagnostic tests. Two sets of primers were shown to specifically amplify sequences either from
any Polymyxa isolate or only from temperate P. graminis isolates and these now provide a sensitive method not only for
detecting the fungus vectors but also for discriminating between the different types.
Extensive molecular comparisons have been made between common and resistance-breaking isolates of BaYMV from
different sites in the UK. Nucleotide sequence differences previously reported between two German isolates proved not
to be strain-related and there were no differences in the coat protein coding region, showing that serological methods (e.g.
monoclonal antibodies) cannot be used to distinguish these isolates. A promising new method, single-strand conformation
polymorphism analysis of fragments of cDNA amplified by RT-PCR, revealed differences between isolates but none was
consistently strain-related. It was therefore decided to proceed with a complete sequencing of the RNA1 genomes of one
common strain and two resistance breaking strain isolates. This is far more work than originally planned and has made it
impossible to achieve one of the final year milestones on time. About 80% of the sequence has been completed and the
isolates have proved to be extremely similar to one another. In only two amino acid positions are there differences which
appear to be associated with virulence differences. These will be tested for consistency using more isolates.
The major components of a test for the viruses and/or their vectors in soil are now in place. These include sensitive PCR
assays for the different isolates of Polymyxa, RT-PCR assays for most of the fungally-transmitted cereal viruses including
two European viruses of wheat, procedures to isolate soil fractions enriched for Polymyxa resting spores and procedures
to extract nucleic acids from resting spores. Experiments have shown that Polymyxa can be detected in the presence of
soil extracts. Some further developmental work is in hand to assess the sensitivity of the test in relation to the inoculum
levels that occur in naturally infested soils.
The operation of Japanese resistance genes that are now being used as alternative sources of resistance to BaMMV and
BaYMV has been investigated. There was no evidence of resistance to the fungus vector and no virus symptoms
developed on plants inoculated a using viruliferous vector isolate. Zoospores and resting spores from resistant plants have
not transmitted virus and it seems likely that the resistance operates against virus multiplication. This form of resistance
is the one that carries least risk of resistance breakdown because there are fewer opportunities for new virus variants to
arise.
Although we now understand something of the viruses and their vectors, more work is needed on the dynamics of
inoculum (in the presence/absence of a susceptible crop; spread of patches; spread of resistance-breaking virus variants)
and its relation to symptom production. Tools developed in this project would enable us to study this and therefore to
predict the speed of spread of new variants and to measure changes in soil populations resulting from changes in crop
practice.
CSG 13 (1/97)
4
Section 5 : Scientific report
INTRODUCTION
Barley mild mosaic (BaMMV) and barley yellow mosaic (BaYMV) viruses, transmitted by the fungus Polymyxa
graminis, pose a long-term threat to winter barley because of their persistence in soil and the variability of the
viruses which makes the stability of genetic resistance (the only effective control measure) uncertain. Similar
viruses of wheat occur in other parts of Europe; they have not so far been confirmed in the UK but could
ultimately become significant (as with Rhizomania of sugar beet). The purpose of this project is to provide
information on the basic biology of the mosaic viruses of cereals, especially BaMMV and BaYMV and their
fungus vector, Polymyxa graminis. This includes studies of vector biology, variability and control; the viruses
and their variability; methods for diagnosis of virus in soil and the nature of cultivar resistance. This report
presents the results of the work done under each of the agreed scientific objectives listed in Section 2. All these
objectives have been met, although objective (3) has involved much more work than originally expected and has
not been met in full; the work to complete it is in hand.
EXPERIMENTAL WORK, RESULTS AND DISCUSSION
(1) Determine the host specificity and properties that control survival, spread and infection, of isolates of
Polymyxa, including their ability to vector viruses and the effects of competition from other soil microbes
Several separate experiments have been used to characterise the biological properties of isolates of Polymxya.
Isolates were obtained from various sources and grown on plant roots in irrigated sand-culture within dedicated
facilities in the Rothamsted greenhouses (Adams, Swaby and Macfarlane, 1986). Roots containing resting
spores of the isolates were air-dried and stored in the laboratory as inoculum for future experiments. The ability
of isolates to infect different hosts was tested by inoculating seedlings with root powder inoculum and growing
the plants in sand culture. The intensity of infection by Polymyxa was assessed microscopically on several root
fragments from each of the replicate pots in the experiment. To test the ability of isolates to transmit viruses,
tillers were removed from plants with virus symptoms and rooted in sterile sand. The fresh roots produced were
then inoculated with the test isolates of Polymyxa and zoospores and resting spores of the fungus subsequently
obtained and used to inoculate susceptible barley seedlings (Adams, Swaby and Jones, 1988). These plants were
maintained in greenhouses or growth rooms under appropriate conditions and examined for virus symptoms.
Plants were also tested by ELISA to confirm the presence and identity of the virus. Transmission experiments
are particularly difficult for BaYMV and only a few successful experiments were done. The results (Table 1)
show that all P. graminis isolates grow best on barley and that most (but not all of them) can also infect wheat.
The majority of isolates tested, including most of those from the UK, were able to transmit the barley mosaic
viruses, but a few isolates (e.g. F36, F53) did not. The results illustrate that variation exists amongst naturallyoccurring isolates of P. graminis but differences in host range in particular often seem to be quantitative and
therefore difficult to define.
To study the effects of competition from other soil microbes and thus to assess the potential for biological
control, soils with widely differing cropping histories were tested for their populations of Polymyxa parasitic on
barley. Tests were done by preparing a dilution series of the test soil with sterile sand and planting single barley
seedlings in small pots of the mixture. Several replicates were done at each dilution and the plants were grown
under standardised watering conditions for 7 weeks at 17°C. Roots were then assessed microscopically for the
presence or absence of Polymyxa resting spores. Populations of Polymyxa in the undiluted soil were estimated
by the most probable number method. Computer simulations and analyses were done to optimise the different
parameters (particularly the dilution ratio and the numbers of replicates) and a standard procedure with a
dilution ratio of 5 and uneven numbers of replicates was adopted (Adams and Welham, 1995). Experiments
were done using the test soils alone and in mixture (50:50) with a standard soil containing a large inoculum of
P. graminis and BaMMV. Results were then examined to determine whether the test soils showed evidence of a
CSG 13 (1/97)
5
File reference: RSC
suppressive effect upon infection in the standard soil. Seven soils were identified with very low (or
undetectable) Polymyxa populations (including long-term fallow, long term pasture and long-term winter wheat
soils) (Table 2) but these all failed to show any evidence of ability to suppress infection by Polymyxa in the
standard soil. The results do not indicate any role for competition by other micro-organisms in affecting the
population levels or activity of P. graminis and suggest that it is unlikely that biological control will prove to be
a useful option
Table 1. Summary of host range and barley mosaic virus transmission experiments
Isolate
F1
F2
F3
F4
F5
F6
F13
F21
F25
F30
F36
F38
F39
F40
F41
F42
F45
F50
F51
F53
F60
Host
Location
Barley
Barley
Barley
Barley
Barley
Barley
Barley
Barley
Poa annua
Barley
Wheat
Barley
Barley
Barley
Sugar-beet
Barley
Barley
Barley
Oats
Wheat
Barley
Streatley, Beds, UK
Streatley, Beds, UK
Swyncombe, Oxon, UK
Harpenden, Herts, UK
Saxmundham, Suffolk, UK
Harpenden, Herts, UK
Box, Avon, UK
Göttingen, Germany
Herts, UK
Streatley, Beds, UK
Prince Edward Island, Canada
Eastleach, Glos, UK
Shanghai, China
Xiaoshan, Zhejiang, China
Barney, Norfolk, UK
Cambs, UK
Chalgrove, Oxon, UK
N. Kincardine, Grampian, UK
Cranbrook, Kent, UK
Ottawa, Canada
Lower Heyford, Oxon, UK
Virus acquisition†
Host range*
Barley
+++
+++
+++
+++
+++
++
+++
+++
++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
++
Wheat
nt
nt
nt
++
++
+
nt
++
++
nt
+++
+
nt
nt
++
+++
+
+++
+++
++
Oat Rye
++
nt
nt
nt
nt
+
nt
+
++
nt
nt
nt
+
nt
+
++
nt
nt
+
++
nt
+
nt
nt
nt
nt
+
nt
nt
+
nt
nt
++
- +++
nt
IRG Sugar-beet
nt
nt
nt
nt
nt
+
nt
nt
+
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
+++
nt
nt
nt
nt
+
nt
BaMMV
+
+
nt
+
+
+
-(1)
nt
+
+
-(5)
+
+
+
nt
-(1)
+
nt
-(1)
-(2)
nt
BaYMV-1 BaYMV-2
+
+
+
* IRG = Italian rye grass; nt = not tested; scored on a scale of none (-) to severe (+++) infection, means of at least 3 replicates.
† nt = not tested; numbers in brackets are numbers of individual experiments in which no transmission occurred.
Table 2. Populations of Polymyxa graminis (most probable numbers of propagules per gram dry soil) estimated in the standard soil
and in various test soils used in suppression experiments
Soil
Standard soil (Continuous winter barley; >15 yr)
Continuous sugar beet (18 yr)
Sugar beet-cereal rotation
Garden soil
Mixed arable rotation
Continuous winter wheat (154 yr)
Pasture (>250 yr)
Continuous spring barley (144 yr)
Fallow (38 yr) after pasture
P. graminis population
26-96*
0
66
0
0
0.02
0
7
0
*, in several experiments
(2) Compare and characterise isolates of Polymyxa by molecular methods and develop diagnostic procedures
based on nucleic acid methods.
The principal approach used has been to amplify a region of ribosomal DNA (rDNA) from fungal isolates using
“universal fungal” primers (White et al., 1990). Because of the difficulties of working with obligate root-infecting
parasites, additional controls have been necessary and DNA has been isolated from fungus zoospores wherever
practicable. A region of about 800bp of rDNA has now been sequenced in 15 isolates of Polymyxa and one isolate
each of Plasmodiophora brassicae, Spongospora subterranea, a Ligniera sp. and the chytrid Olpidium brassicae.
6
File reference: RSC
This region consists of about 330 bp at the 3' end of the 18S-like gene, the 5.8S DNA and the two internal
transcribed spacers. The results showed that isolates of P. graminis could be allocated to one of three subgroups
(only two of which are represented by temperate isolates). All the isolates in each group had identical sequences
and the groups correspond to those that had earlier been identified by RFLP analysis (Ward et al., 1994).
Comparison with the biological experiments suggests that the “Type II” subgroup isolates were those that did not
transmit BaMMV. P. betae sequences were identical to one another and were sufficiently different to the P.
graminis ones to justify their separation as a distinct species. In phylogenetic analyses of the NS7/NS8 region, the
Ligniera and Spongospora isolates grouped with the other plasmodiophorids and the Olpidium isolate with the
Mycota (Fig. 1). The plasmodiophorids appeared as a distinct group and were not closely related to any other
eukaryotes (including a range of fungi and protozoa). Subsequent to this work, a fourth P. graminis subgroup, most
closely related to subgroup II, has been identified for isolates from Colombia transmitting rice stripe necrosis virus.
Physarum
Giardia
Polymyxa betae
100
Polymyxa graminis II
78
Polymyxa graminis I
99
100
plasmodiophorids
Polymyxa graminis-IPCV
Plasmodiophora UK
100
Plasmodiophora USA
Basidiobolus
Zygomycetes
65
Olpidium
77
Chytridium
Chytridiomycetes
fungi
94
66
Spizellomyces
100
Pichia
65
Phanerochaete
100
Rhizoctonia
Phytophthora
100
Lagenidium
*
53
Oomycetidae
Achlya
68
straminipiles
Pseudo-nitzschia
100
Aulacoseria
71
38
Diatomea
Eucampia
Stylonychia
Sarcocystis
44
protoctists
100
44
Apicomplexa
Neospora
91
Toxoplasma
Stylonema
89
Leptobryum
red and green plants
0.1
Fig. 1. The phylogenetic tree of the NS7-NS8 regions obtained using NEIGHBOR analysis and displayed as a Phylogram from
VIEWTREE. The values at the forks indicate the number of times out of 100 trees that this grouping occurred after bootstrapping the data.
Dotted lines show largely unsupported branches (bootstrap values <70%). There is no bootstrap value for the asterisked fork as this
grouping did not appear in the consensus tree produced after bootstrap analysis. Instead the Phytophthora and Lagenidium grouped
together with a bootstrap value of 55%. With this exception, the tree obtained from the original data set and the consensus tree obtained
after bootstrapping were identical. The horizontal lines are to the scale shown in the bottom left of the figure (substitutions per base).
The DNA sequences were then used to design primers for specific amplification of either Polymyxa or P. graminis
DNA. The Polymyxa-specific primers (Pxfwd1/Pxrev7) only amplified DNA from Polymyxa isolates, not from any
of the other samples, and the P. graminis-specific primers (Pgfwd2/Pxrev7) amplified DNA only from P. graminis
type I and II isolates and not from IPCV-P. graminis (isolates from India transmitting Indian peanut clump virus),
7
File reference: RSC
P. betae nor any of the other samples. Other organisms that tested negative with both sets of primers included other
plasmodiophorids (Plasmodiophora brassicae, Spongospora subterranea and Ligniera sp.), other zoosporic fungi
(Olpidium brassicae and three Phytophthora species), a range of other soil-inhabiting and root-infecting fungi and
appropriate uninfected plant controls (barley, wheat, sugar beet). The primers worked well with DNA extracted
from zoospores, resting spores and infected plant roots. The products of amplification using primers Pxfwd1 +
Pxrev7 on isolates of Polymyxa graminis types I and II and P. betae could all be distinguished from one another by
their size on 1.2% agarose gels, but the differences were clearer using gels containing 2% Nusieve agarose + 1%
standard agarose. This PCR is therefore not only useful in specifically detecting Polymyxa species, but also allows
discrimination between the species and RFLP groups. It was also possible to distinguish the Polymyxa graminis
types I and II in the same way using primers Pgfwd2 + Pxrev7. These primers therefore provide a sensitive method
not only for detecting the fungus vectors but also for discriminating between the different groups.
(3) Characterise, in terms of biological and molecular properties, variant isolates of BaYMV and develop
methods for their diagnosis using nucleic acid based methods.
In Britain and other parts of Europe, a resistance-breaking strain of barley yellow mosaic virus, usually known
as BaYMV-2 has been becoming gradually more frequent (Huth, 1991; Adams, 1998). To determine the
difference between common and resistance-breaking isolates and to develop diagnostic methods that would
distinguish them, several isolates of BaYMV were obtained from different sites in the UK, including some of
the resistance-breaking strain and some that were not. These were examined by RT-PCR, restriction mapping
and sequencing of selected parts of the virus genome: the 5' terminal region, part of the NIa coding region and
the coat protein coding region on RNA 1 and an area at the N-terminus of the 70 kDa protein coding region on
RNA 2 (see Fig. 2). These regions were chosen because of their variability and because experiments in Germany
had suggested that the difference between the common strain and BaYMV-2 might lie within the RNA 2 region.
The sequences obtained differed from those previously reported for a BaYMV isolate from Japan and for two
German isolates, one of which was of the BaYMV-2 strain. There were no strain-specific amino acid differences
and the few, non-consecutive, nucleotide differences detected were probably not significant and were
insufficient to develop a rapid diagnostic test to distinguish BaYMV-2 from other isolates. Restriction mapping
of RNA 2 cDNA again showed no consistent strain-related differences. It was therefore concluded that the
differences previously reported between the two German isolates (Bendiek et al., 1993) were not strain-related.
The lack of difference in the coat protein coding region means that monoclonal antibodies cannot be used to
distinguish these isolates.
Further attempts to identify differences between the two strains used a promising new method, single-strand
conformation polymorphism (SSCP) analysis of fragments of cDNA amplified by RT-PCR. In this technique,
double-stranded DNA from the RT-PCR reaction is denatured to single-stranded DNA and each strand then
assumes a specific folded conformation based on internal base-pairing, which in turn depends on its sequence.
These conformers are separated by polyacrylamide gel electrophoresis under non-denaturing conditions. The
migration of the conformer depends on its folded structure, and thus point mutations or polymorphisms in DNA
fragments can cause detectable mobility shifts. DNA fragments were visualised by silver staining. PCR-SSCP is
claimed to be a rapid, sensitive, non-radioisotopic method for detection of both known and unknown mutations
or polymorphisms in products of PCR. It therefore seemed promising for the detection of unknown differences
between virus strains. Primers were designed to amplify fragments, typically of around 800 nucleotides,
covering the whole of both RNA1 and RNA2 from one common strain isolate and two resistance breaking
isolates of UK BaYMV. Where patterns that appeared to be related to resistance breaking were identified, that
region was checked with three further isolates (one common strain isolate and two resistance breaking). Isolates
differed in their SSCP patterns in several regions, but in no case was the pattern able to distinguish between
common and resistance-breaking strains. In regions where the nucleotide sequences of UK isolates had been
determined, there was no simple relationship between numbers of nucleotide differences and SSCP patterns:
differences of only 2 or 3 nucleotides gave different SSCP patterns, whereas differences of as many as 29
nucleotides did not. It was concluded that although SSCP analysis has some potential as a rapid and sensitive
tool for distinguishing virus isolates, differences detected do not necessarily relate to biological properties and
8
File reference: RSC
the results are highly dependent on gel conditions.
5'
3'
RNA 1
unknown
cell-to-cell movement
unknown
particle protease
assembly (protein
processing)
unknown
polymerase
(replication)
virus coat
protein
RNA 2
protease
(protein
processing)
fungus transmission
Fig. 2. Diagram showing the genome organisation of barley yellow mosaic virus. The boxes represent the predicted
protein products and their probable function is indicated below. Cross-hatched boxes are the regions initially targeted for
restriction analysis and sequencing and the regions currently sequenced in at least three UK isolates are shown by the
arrow.
Because of the failure of the earlier, targeted, approach to identify the significant differences between the two
BaYMV strains, it was decided to proceed with a complete sequencing of the genomes of the two types of
BaYMV. This is a much greater effort than originally planned for this project but substantial progress has now
been made with sequencing RNA1 of one common strain and two resistance breaking strain isolates. RNA1 was
chosen because current knowledge of bymovirus gene function makes it likely that most of the major virulence
determinants will be found on this genome segment. At the time of writing, sequences of over 7,000 bases have
been determined from each of the three isolates and the sequences from RNA1 are about 85% complete (Fig. 2).
Analysis of the data collected so far shows that the isolates are extremely similar to one another and that where
differences occur, they are not usually linked to resistance-breaking or are silent (i.e. produce no changes to the
sequence of the predicted amino acid product). In only two amino acid positions are there differences which
appear to be associated with virulence differences (polyprotein amino acid numbers 1156 and 1767) but these
will need to be tested for consistency using more isolates when the complete RNA1 sequence is available.
Until the complete sequence (at least of RNA1) is determined, it is not possible to design a PCR-based test to
distinguish the two strains and thus the milestone set for the end of the project has not been reached. However,
because of the importance of this work, it is now continuing with core BBSRC funding and I am confident that
it will be completed by mid-1999 and we can then begin screening samples that were collected for this purpose
in Winter 1998 and new samples we expect to receive during the first months of 1999. Results will be reported
to MAFF under the new commissioned project which is expected to start in April 1999.
(4) Develop methods, based on current and future immunology and nucleic acid assays, for diagnosing
fungally-transmitted cereal viruses and their vector in soil.
We have developed sensitive immunological tests for each of the fungally-transmitted cereal viruses. These tests
can be used in combination with bioassays of soils with test seedlings to determine whether a soil is infested
with virus but this procedure is rather slow and labour-intensive for routine use. There would be advantages in a
test that could be used directly in the laboratory on soil samples, but serological assays have not proved suitable
9
File reference: RSC
for use with soil samples because serological reactions are inhibited by soil components. Experiments have
therefore concentrated on the use of nucleic acid based assays.
The major components of a test for the viruses and/or their vectors in soil are now in place:
(a) Sensitive PCR assays for the different isolates of Polymyxa have been developed (see section 2 above).
(b) RT-PCR assays have been developed for most of the fungally-transmitted cereal viruses. Assays for the two
barley viruses, BaMMV and BaYMV have been developed which both amplify a DNA fragment from the 3' end
of RNA-1, including the capsid protein region. Primers M3 and M4 (Table 3) give an 899 bp DNA fragment for
BaMMV and primers 4800EP and 4799EP give a BaYMV fragment of 1018 bp. These assays were able to
detect less than 1 pg plM7 DNA (a plasmid containing 6.3 kb cDNA of BaMMV RNA-1) and 0.1 pg ply29
DNA (a plasmid containing a 6 kb cDNA insert of BaYMV RNA-1). In experiments with two European viruses
of wheat (WSSMV and a furovirus similar to SBWMV which we are naming European wheat mosaic virus,
EWMV) we have developed a single two-step RT-PCR that is able to detect both viruses in the same
experiment. This uses a combination of the primer sets Fs5/Rs5 and Fw4/Rw4 (Table 3) and results in
fragments of 457 (WSSMV) and 718 (EWMV) bp. The test has proved sensitive and reliable in an extensive
comparison with ELISA using 100 infected wheat samples from France. We also have RT-PCR protocols that
are able to detect OGSV from oats but so far we have not succeeded in getting sequence data from OMV to use
in such assays.
Table 3. Primer sets developed for the detection of the fungally-transmitted viruses of barley and wheat
Primer
Sense*
M3
+
M4
BaYMV
4800EP
+
4799EP
EWMV
Fs5
+
Rs5
WSSMV
Fw4
+
Rw4
*
+, forward primer; -, reverse primer
Virus
BaMMV
Sequence of primer (5' to 3')
ACA GAG CAC GAG GAG
GCA TGA GAG ATC TAC
GGC TGC AAG CAG CTG
GCC AAA GGC ATA TGG
ATG ATA AAT TTC AGG
TAC TTC GAC CCA AAC
AAG GAA ATA AAA TAC
TCA TAC CCG ACT CTT
GAA
CGG
ATC
ATC
GCA
CGT
CGC
CCA
Size (bp)
899
CTC
ATA
GCG
CAT
CCC
GCA
TCA CTG
GAA CCG
AC
TT
AG
C
1018
718
457
(c) Procedures to isolate soil fractions enriched for Polymyxa resting spores have been developed. Two methods
for separation of soil fractions have been investigated. In the first of these, dispersed soil samples are
fractionated by layering onto a Percoll-sucrose gradient and gentle centrifugation. This method is claimed to
separate particles on the basis of their density in an environment that does not exert disruptive osmotic
pressures. It has proved difficult to get reproducible and precise banding of introduced Polymyxa spores within
such gradients and the method is limited in the quantities of soil that can be processed. In most experiments we
have therefore used aqueous two-phase partitioning (A2PP). This method also uses dispersed soil samples but
they are separated on the basis of hydrophobicity between two different aqueous solutions of high molecular
weight polymers (Smith and Stribley, 1994). We have used dextran (MW about 500,000) and polyethylene
glycol (PEG; MW 8,000) solutions and run experiments to test the effects of different polymer concentrations
and different types and strength of aqueous buffer. A protocol has been developed that uses 4.5% dextran and
4% PEG in a 0.1M Sorensen phosphate buffer pH 7.4. Most of the soil goes into the bottom phase, while the top
phase, which has the majority of the Polymyxa spores, is then further purified by centrifugation over a Percollsucrose cushion. Over 50% of introduced Polymyxa spores can be routinely recovered by this procedure.
(d) Procedures have been developed to extract nucleic acids from resting spores and experiments have shown
that Polymyxa can be detected in the presence of soil extracts.
All the major components for a test are now in place but some further developmental work is needed to assess
the sensitivity of the test in relation to the inoculum levels that occur in naturally infested soils. These
experiments are in hand.
10
File reference: RSC
(5) To determine the basis of resistance to mosaic viruses in Japanese cultivars in current use by UK plant
breeders
Until recently, UK and European plant breeders relied exclusively on a single recessive gene (ym4) when
breeding for resistance to BaYMV and BaMMV. Because BaYMV-2 is virulent on such cultivars, some
Japanese resistance genes are being investigated and used as alternative sources of resistance. We already know
that the ym4 gene operates against the virus (and not the fungus vector) and that it operates at a very early stage
after inoculation, preventing virus multiplication and rendering the plants immune (Adams, Jones and Swaby,
1987). To obtain comparable information for the Japanese resistance genes, barley breeding lines incorporating
one or more such genes were obtained from Advanta seeds. These were inoculated with P. graminis resting
spores from an isolate transmitting BaMMV and grown in sand culture in the greenhouses. Large numbers of P.
graminis zoospores were obtained from all plants tested showing that there was no evidence of resistance to the
fungus vector. As expected, no virus symptoms developed on the inoculated plants (except in the susceptible
control cultivar). Zoospores and resting spores from plants with Japanese resistance genes were then inoculated
to seedlings of a standard virus-susceptible barley cultivar to determine whether they were carrying virus. These
experiments are still continuing, but to date no virus has been transmitted from these plants and it therefore
seems likely that the resistance operates in a manner similar to the ym4 gene, i.e. against virus multiplication.
This form of resistance is the one that carries least risk of resistance breakdown because there are fewer
opportunities for new virus variants to arise.
POSSIBLE FUTURE WORK
Much of the future strategy against barley mosaic viruses depends upon the efforts of plant breeders to produce
resistant cultivars that can match susceptible cultivars for quality and yield potential. However, the resistance
will be challenged by the variability of the viruses and the selection pressure will be increased by any trends
towards more, or more intensive, cereal production as a result of CAP reform. Although we now understand
something of the viruses and their vectors, the dynamics of inoculum (in the presence/absence of a susceptible
crop; spread of patches; spread of resistance-breaking virus variants) and its relation to symptom production is
not understood. Tools we are currently using and developing will enable us to study this and therefore to predict
the speed of spread of new variants and to measure changes in soil populations resulting from changes in crop
practice (including new chemicals to control root diseases which might have a direct or indirect effect on
Polymyxa). Results would have a direct bearing on the stability of existing or new resistance genes, a matter of
concern to plant breeders and farmers alike. Such studies would also provide information to help shape a
response to any proven introduction or detection of the fungally-transmitted viruses of winter wheat in the UK.
It is hoped that such work will feature in a new MAFF Commission starting in April 1999.
LITERATURE CITED
ADAMS, M. J. (1998). Fungally-transmitted mosaic viruses of barley. UK Cereal Pathogen Virulence Survey,
1997 Annual Report, pp. 83-85. Cambridge: UK Cereal Pathogen Virulence Survey Committee.
ADAMS, M. J., JONES, P. & SWABY, A. G. (1987). The effect of cultivar used as host for Polymyxa graminis
on the multiplication and transmission of barley yellow mosaic virus (BaYMV). Annals of Applied Biology 110,
321-327.
ADAMS, M. J., SWABY, A. G. & JONES, P. (1988). Confirmation of the transmission of barley yellow mosaic
virus (BaYMV) by the fungus Polymyxa graminis. Annals of Applied Biology 112, 133-141.
ADAMS, M. J., SWABY, A. G. & MACFARLANE, I. (1986). The susceptibility of barley cultivars to barley
yellow mosaic virus (BaYMV) and its fungal vector, Polymyxa graminis. Annals of Applied Biology 109, 561-572.
11
File reference: RSC
ADAMS, M. J. & WELHAM, S. J. (1995). Use of the most probable number technique to quantify soil-borne
plant pathogens. Annals of Applied Biology 126, 181-196.
BENDIEK, J., DAVIDSON, A., SCHULTZE, S. C., SCHELL, J. & STEINBISS, H.-H. (1993). Identification and
classification of a resistance breaking strain of barley yellow mosaic virus. Annals of Applied Biology 122, 481491.
HUTH, W. (1991). Verbreitung der Gelbmosaikviren BaYMV, BaMMV und BaYMV-2 und Screening von
Gerstensorten auf Resistenz gegenüber BaYMV-2. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes
(Stuttgart) 43, 233-237.
SMITH, N. C. & STRIBLEY, D. P. (1994). A new approach to direct extraction of microorganisms from soil. In:
K. Ritz, J. Dighton & K. E. Giller. Beyond the Biomass. Chichester: John Wiley & Sons, 49-55.
WARD, E., ADAMS, M. J., MUTASA, E. S., COLLIER, C. R. & ASHER, M. J. C. (1994). Characterization of
Polymyxa species by restriction analysis of PCR-amplified ribosomal DNA. Plant Pathology 43, 872-877.
WHITE, T. J., BRUNS, T., LEE, S. & TAYLOR, J. (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In PCR Protocols. A Guide to Methods and Applications (ed. Innis, M.
A., Gelfand, D. H., Sninsky, J. J. & White, T. J.), pp. 315-322. Academic Press: San Diego, USA.
12