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Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world Q7
Wolfgang GRASSE, Otmar SPRING* Institute of Botany, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany
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article info
abstract
Article history:
Plasmopara halstedii virus (PhV) is a ss(þ)RNA virus that exclusively occurs in the sunflower
Received 16 September 2014
downy mildew pathogen P. halstedii, a biotrophic oomycete of severe economic impact. The
Received in revised form
origin of the virus and its genomic variability in host populations from different geographic
28 November 2014
regions is unknown.
Accepted 8 December 2014
Sporangia of 128 samples of P. halstedii from five continents, collected over the past 40 y, were screened for presence of PhV using PCR-based molecular tools. PhV RNA was found in over 90 % of all P. halstedii isolates with no correlation to any specific
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Keywords:
geographic origin or pathotype of its host. Sequence analyses of the open reading frames
Genome diversity
(ORFs) were performed to show the genetic diversity of the virus. Only 18 single nucleotide
Mycovirus
polymorphisms (SNPs) were found in the 3873 sequenced nucleotides of the two ORFs and
Oomycetes
these SNPs had no recognizable effect on the encoded proteins. In the 398 nucleotides of
ss(þ)RNA
the untranslated regions (UTRs) of the RNA 2 strand eight additional SNPs and one short
Sunflower downy mildew
deletion was found. Modelling experiments revealed no effects of these variations on the secondary structure of the RNA. The results showed the presence of PhV in P. halstedii isolates of global origin and the existence of the virus since more than 40 y. The virus genome revealed a surprisingly low variation in both coding and noncoding parts and none of the differences was correlated with the host pathotype or geographic populations of the oomycete. ª 2014 Published by Elsevier Ltd on behalf of The British Mycological Society.
Introduction Plasmopara halstedii virus (PhV) is one of the rare characterized viruses in obligate biotrophic Oomycetes (Yokoi et al. 1999, 2003; Heller-Dohmen et al. 2011). PhV is a ss(þ)RNA virus with two RNA segments encoding for the RNA depending RNA polymerase (RNA strand 1, ORF 2745 nucleotides) and the coat protein (RNA strand 2, ORF 1128 nucleotides), respectively. Its virions are isometric, measure approximately 37 nm
in diameter, and were found in all developmental stages of its exclusive host P. halstedii, the downy mildew pathogen of sunflower (Heller-Dohmen et al. 2008, 2011). PhV occurs in the cytoplasm of hyphae, haustoria, and sporangia of its host and may reach very high numbers as was shown in transmission electron microscopy studies by Heller-Dohmen et al. (2008). A hypovirulence effect of virus infection on the aggressiveness (intensity and speed of sporulation), but not on the pathogenicity (infectivity to differentiating host genotypes) of the
* Corresponding author. Tel.: þ49 711 459 23811; fax: þ49 711 459 23355. E-mail address: [email protected] (O. Spring). http://dx.doi.org/10.1016/j.funbio.2014.12.004 1878-6146/ª 2014 Published by Elsevier Ltd on behalf of The British Mycological Society.
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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oomycete was recently shown by infection experiments with two genetically homogenous strains with and without virus (Grasse et al. 2013). While P. halstedii was reported to occur in all major areas of sunflower cultivation except for Australia (Gulya et al. 1997; Gulya 2007; Viranyi & Spring 2011), the distribution of PhV in samples of P. halstedii from different geographical origins has been investigated only in a very limited number of samples (Heller-Dohmen et al. 2008, 2011). In particular the genomic variation of PvH from different origins has not been investigated in detail to date. This is similar to most known mycoviruses and is most likely due to the lack of suitable sample material. Only recently, a study was published on the occurrence of partitiviruses in seven Heterobasidion species where it was found that only 5 % of the fungi were virusinfected. Three different strains of the HetRV virus were found in ten samples, but no detail on the sequence variation in the virus genome was provided (Vainio et al. 2011). Our intensive collecting of P. halstedii isolates in Central Europe and South America as well as access to sample material from East Europe (F. Viranyi, Szent Istvan University, € do € llo € , Hungary), Africa, Asia, and North America (T.J. Gulya, Go USDA Fargo, U.S.A.) enabled us to screen for the occurrence of PhV in samples from the past 40 y. We here report on a PCRbased method for the detection of the ss(þ)RNA virus in sporangia of the oomycete. Moreover, we investigated the genetic diversity of PhV by sequence comparison of the open reading frames (ORFs) encoding for the capsid protein (CP) and the RNA depending RNA polymerase (RdRp) and by comparing the untranslated regions (UTRs) of the genome.
Methods Origin of the used Plasmopara halstedii isolates In this study, 128 Plasmopara halstedii isolates collected within the past 40 y from 17 countries on five continents were screened for the presence of PhV. The sample material either consisted of freshly harvested sporangia, sporangia which had been stored for up to ca. 40 y in the freezer, or downy mildew infected sunflower leaves with visible sporulation from herbarized specimens. The origin, year collected, condition, and provider of samples are listed in Table 1.
Nucleic acid extraction The total RNA was extracted using the BioRAD Aurum RNA Extraction Kit (Bio-Rad Laboratories, Hercules, US-CA). In case of herbarized samples, polyclearAT (1 mg/10 mg sample) was added to bind residual of phenolic substances. The RNA was transcribed into cDNA according to the manufacturer’s protocol using RevertAid reverse Transcriptase (Thermo Fisher Scientific; US-MA) and a random hexamer primer.
PCR-based virus screening The screening of the PhV samples was performed by PCR using four primer pairs specific for short sequences of both strands of the virus genome (Table 2). The primers
W. Grasse, O. Spring
PHV_pol_F1/R1 and PHV_pol_F2/R2 were used to amplify a 294 and a 390 bp fragment of the RdRp gene, respectively. The primers 2f3/PHV_WG_R1 and PHV_WG_F1/R2 were employed to amplify a 225 bp and a 324 bp fragment of the coat protein gene. PCRs were performed using Genaxxon RedTaq Mix (Genaxxon, Ulm, Germany). PCR amplifications were performed on a peqSTAR thermocycler (Peqlab, Erlangen, Germany) using the following cycling protocol: initial denaturation at 94 C for 3 min followed by 40 cycles with denaturation at 94 C for 20 s, annealing at 60 C for 20 s and elongation at 72 C for 20 s; the final extension was carried out at 72 C for 5 min. The PCR products were analysed on 2 % Agarose gels (1 TBE buffer, ethidium bromide staining). Samples were considered to be virus-free when they had been tested negative in two independent experiments.
Amplification and sequencing of cDNA PCR of cDNA amplifications were performed on a peqSTAR thermocycler (Peqlab, Erlangen, Germany) using the Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Karlsruhe) and the following cycling protocol: initial denaturation at 98 C for 3 min followed by 40 cycles with denaturation at 98 C for 20 s, annealing at 55e60 C for 20 s and elongation at 72 C for 120 s; the final extension was carried out at 72 C for 10 min. The PCR products were analysed on 1.5 % Agarose gels (1 TBE buffer, ethidium bromide staining). PCR products of the ORFs and the UTRs generated with specific primers (for used primers see Table 2) were sequenced commercially (Macrogen, Amsterdam, Netherlands). For sequence comparison the sequences of PhV coat HM453718 and RdRp HM453713 of sample #24 (a virus isolate from France 1999) published by Heller-Dohmen et al. (2011) were used as reference. The alignment was done with clustalW algorithm from the software BioEdit (Ibis Bioscience, USA). The mean distance was estimated with the program MEGA5 (Tamura et al. 2011).
Modelling of 30 UTR structure The secondary RNA structure of the 30 UTR was performed with the minimal free energy algorithm of the Vienna RNA webserver (Zucker & Stiegler 1981; Hofacker et al. 1994). For the visualization of the secondary RNA structure the software PseudoViewer 2.5 was used (Han & Byun 2003).
Results Occurrence of PhV in Plasmopara halstedii samples of different continents PhV viral RNA was successfully extracted and translated into cDNA from Plasmopara halstedii sporangium samples which were collected up to 40 y ago. The screening with four specific primer pairs (Table 1) for short sequences of both RNA strands showed the presence of PhV in almost all cases, no matter whether freeze-stored sporangia, freshly harvested sporangia or sunflower leaves with visible sporulation from old herbarized plant specimens were used. Out of 128 samples of different
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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Table 1 e Origin and collection dates of P. halstedii samples used for the PCR-based PhV screening. Samples used for sequencing are highlighted in dark grey; samples analysed by transmission electron microscopy in the previous study of Heller-Dohmen et al. (2008) are marked in light grey. Sample #
Provided
a
b
c
44
OS
Germany
2003d
hl
+
45
OS
Germany
2003e
hl
+
46
OS
Germany
2004a
hl
+
47
OS
Germany
2004b
hl
+
48
OS
Germany
2004c
hl
+
Country
Date
Source
PhV
49
OS
Germany
2004d
hl
+
1
TG
Argentina
1999
fs
+
50
OS
Germany
2004e
hl
+
2
TG
Argentina
2000a
fs
+
51
OS
Germany
2005a
hl
+
3
TG
Argentina
2000b
fs
+
52
OS
Germany
2005b
hl
+
4
OS
Argentina
2003a
hl
+
53
OS
Germany
2005c
hl
+
5
OS
Argentina
2003b
hl
-
54
OS
Germany
2005d
hl
+
6
OS
Austria
1999a
hl
+
55
OS
Germany
2005e
hl
+
7
OS
Austria
1999b
hl
+
56
OS
Germany
2008a
hl
+
8
TG
Bulgaria
1990
fs
+
57
OS
Germany
2008b
hl
+
9
TG
Bulgaria
1999
fs
+
58
OS
Germany
2008c
hl
+
10
TG
Canada
1991
fs
+
59
OS
Germany
2010a
s
-
11
TG
Canada
1992
fs
+
60
OS
Germany
2010b
s
+
12
TG
Canada
1995
fs
+
61
OS
Germany
2010c
s
-
13
TG
Canada
1996a
fs
+
62
OS
Germany
2010d
s
+
14
TG
Canada
1996b
fs
+
63
OS
Germany
2010e
s
-
15
TG
Canada
1998
fs
+
64
OS
Germany
2011a
s
+
16
OS
Canada
2001
hl
+
65
OS
Germany
2011b
s
-
17
OS
Canada
2002
hl
-
66
OS
Germany
2011c
s
-
18
FV
China
1987
hl
+
67
OS
Germany
2012a
s
+
19
TG
China
1992
fs
+
68
OS
Germany
2012b
s
+
20
TG
France
1988
fs
-
69
OS
Germany
2012c
s
+
21
TG
France
1989
fs
+
70
OS
Germany
2012d
s
+
22
TG
France
1991
fs
+
71
OS
Germany
2012e
s
-
23
TG
France
1998
fs
+
72
OS
Germany
2012f
s
+
24
OS
France
1999
hl
+
73
OS
Germany
2012g
s
+
25
TG
France
2000a
fs
+
74
OS
Germany
2012h
s
+
26
OS
France
2000b
hl
+
75
OS
Germany
2012i
s
+
27
OS
France
2000c
hl
+
76
OS
Germany
2012j
s
+
28
OS
France
2000d
hl
+
77
FV
Hungary
1972
hl
+
29
OS
France
2000e
hl
+
78
FV
Hungary
1974
hl
+
30
TG
Germany
1992
fs
+
79
FV
Hungary
1976
hl
+
31
OS
Germany
1993
hl
+
80
FV
Hungary
1984a
hl
+
32
OS
Germany
1996
hl
+
81
FV
Hungary
1984b
hl
+
33
OS
Germany
1997a
hl
+
82
TG
Hungary
1988
fs
+
34
OS
Germany
1997b
hl
+
83
FV
Hungary
1989
hl
+
35
OS
Germany
1998a
hl
+
84
TG
Hungary
1990a
fs
+
36
OS
Germany
1998b
hl
+
85
TG
Hungary
1990b
fs
+
37
OS
Germany
2000
hl
+
86
TG
Hungary
1991
fs
+
38
OS
Germany
2001a
hl
+
87
TG
Hungary
1992
fs
+
39
OS
Germany
2001b
hl
+
88
TG
Hungary
1993
fs
+
40
OS
Germany
2001c
hl
-
89
TG
Hungary
1995a
fs
+
41
OS
Germany
2003a
hl
+
90
FV
Hungary
1995b
hl
+
42
OS
Germany
2003b
hl
+
43
OS
Germany
2003c
hl
+
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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W. Grasse, O. Spring
91
OS
Hungary
2001a
hl
+
92
OS
Hungary
2001b
hl
+
93
TG
India
1995
fs
+
94
TG
Italy
1998
fs
+
95
TG
Morocco
1997a
fs
+
96
TG
Morocco
1997b
fs
+
97
TG
Morocco
1997c
fs
+
98
TG
Morocco
1998
fs
+
99
OS
Slovakia
1999
hl
+
100
TG
South Africa
1996a
fs
+
101
TG
South Africa
1996b
fs
+
102
TG
South Africa
1996c
fs
+
103
TG
South Africa
1997a
fs
+
104
TG
South Africa
1997b
fs
+
105
TG
Spain
1986
fs
+
106
TG
Spain
1994
fs
+
107
TG
Spain
1997
fs
+
108
TG
Spain
1998
fs
+
109
TG
Spain
1999
fs
+
110
OS
Switzerland
2011
hl
-
111
TG
Turkey
1996a
fs
+
112
TG
Turkey
1996b
fs
+
113
TG
Turkey
1997a
fs
+
114
TG
Turkey
1997b
fs
+
115
TG
Turkey
1997c
fs
+
116
TG
USA
1985a
fs
+
117
TG
USA
1985b
fs
+
118
TG
USA
1988
fs
+
119
TG
USA
1989
fs
+
120
TG
USA
1991a
fs
+
121
TG
USA
1991b
fs
+
122
TG
USA
1992
fs
+
123
OS
USA
1993
hl
+
124
TG
USA
1994
fs
+
125
OS
USA
1997
hl
+
126
TG
USA
1998
fs
+
127
TG
USA
1999
fs
+
128
OS
USA
2007
hl
-
age and origin, 117 tested positive which indicates that more than 90 % of the P. halstedii isolates harboured the virus genome (Fig 1; Table 2). In all positive samples, amplification products of both RNA strands were found. No correlation was found between the geographic origin of samples, and presence or absence of PhV sequences. Virus-free samples were found among the samples from North America (2 out of 21), South America (1 out of 5), and Europe (8 out of 91), whereas samples from Africa (9) and Asia (3) all contained PhV sequences. Negative PCR results occurred in samples from fresh sporangia as well as from herbarized specimens. With respect to the seemingly higher rate of virus-free samples in Germany (6 out of 45) and Switzerland (1 out of 1) it is noteworthy that the majority of collections in these countries derived from small fields of cutting sunflowers where mostly sunflower seeds with unknown breeding history were cultivated and no chemical plant protection against downy mildew was employed. The occurrence of PhV was not correlated with the sampling date of the P. halstedii isolate. The oldest sample containing PhV sequences was
collected in 1972 in Hungary, while the oldest virus-free sample was from France collected in 1988.
Sequence variation in the two ORFs Short PCR fragments of the two coding regions for the RdRp and CP of PhV could readily be amplified for all isolates. In contrast, longer amplicons were sometimes difficult to obtain, possibly because the RNA might have been partly degraded over the time of storage. Sequencing of the complete ORFs (2745 nucleotides for RdRp; 1128 nucleotides for CP) was achieved for 22 samples from 13 countries. Alignments of the 3873 bp complete ORFs showed five positions in which single nucleotide polymorphisms (SNPs) consistently occurred. In addition, 13 SNPs only occurred in sequences of one or two samples (position numbers as given in Table 2; for base identity see Additional File 1, 2). Compared with reference genome of PhV reported for sample #24 (GenBank PhV coat HM453718 and RdRp HM453713) the five consistent positions were nt. 1242 (A/G exchange) and nt. 2443 (A/G) in case of the RdRp (RNA1), and position nt. 372 (C/ T), nt. 700 (T/G) and nt. 888 (C/T) in case of the coat protein (RNA2), respectively (Table 2). These five SNPs lead to amino acid exchanges in only three cases, because SNP RNA1 1242 and SNP RNA2 700 were silent mutations. SNP RNA1 2443 accounted for an alanine/threonine exchange and occurred in 18 of the 22 samples. The SNPs RNA2 372 and 888 caused an alanine/valine and threonine/isoleucine exchange in the coat protein, respectively. The former was found in 15, the latter occurred in five of the 22 samples. The 13 additional SNPs led to 11 amino acid changes in the RdRp (nt. 1972 alanine/threonine, nt. 1973 alanine/valine, nt. 2173 methionine/valine, nt. 2467 phenylalanine/valine, nt. 2583 aspartate/glutamate and nt. 2598 histidine/glutamine) and the coat protein (nt. 400 isoleucine/ methionine, nt. 542 valine/isoleucine, nt. 674 glutamate/glutamine, nt. 679 asparagine/lysine, and nt. 728 serine/threonine). The highest accumulation of SNPs (8) compared to the reference sequences appeared in an Argentinian sample from 1999 (#1). None of the sequenced samples was completely identical with the reference. The lowest number of SNPs (1) in the ORFs was found in two samples from Canada (#13, 1996a) and Germany (#51, 2005a). In no case we observed a correlation of SNPs with the origin or age of the samples and no coincidence between nucleotide exchanges in the RdRp versus CP became visible. A group of seven samples (indicated by sidebar in Table 2) showed identical ORFs, but their origin from three continents and sampling dates over a period of 12 y does not imply an internal relationship. In four samples (Bulgaria 1999; USA 1999; Argentina 2000b; Germany 2003) mixed populations of two different PhV genotypes was detected. Within the complete ORFs the genetic diversity of the 22 samples was extremely low with a mean of distance of 0.001 for the RdRp and 0.002 for the CP.
Sequence variation in the UTRs The presumable amplicons of the UTRs of the RNA1 strand (RdRp) were too short (18 nt; 30 nt) for sequence comparison outside of the primer binding sites. Therefore, we focused on the UTRs of RNA2 (CP) which consisted of 166 nt and 234 nt for the 50 and 30 end, respectively. Complete sequences of these regions were obtained from 12 samples out of 9
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
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5
Table 2 e Position of SNPs in ORFs of PhV isolates. Sample #24 (France 1999) served as reference genome. SNPs found in only one or two samples are indicated by the nt. position. Samples identical in sequence are marked by side bar. Sample
Country
RdRpa
Date
1242 A/G 2443 G/A 24 43 51 9 58 89 13 2 3 11 93 127 21 94 95 100 105 122 124 113 1 8
c
France Germany Germany Bulgaria Germany Hungary Canada Argentina Argentina Canada India USA France Italy Morocco S. Africa Spain USA USA Turkey Argentina Bulgaria
1999 2003c 2005a 1999 2008c 1995a 1996a 2000a 2000b 1992 1995 1999 1989 1998 1997a 1996 1986 1992 1994 1997a 1999 1990
A A/G A A A A A A A/G G G G G G G G G G G G G G
G G G G A A A A A A A A A A A A A A A A A A
CPb
Additional SNPs
372 T/C
700 T/G
888 C/T
e e e 2598 (T/A) e e e 1972 (G/A); 1973 (C/T) 2467 (T/G) 888 (C/A); 2173 (A/G) e e e e e e e e e e 1973 (C/T); 2583 (C/A) e
T C C C C C T T T C C C C C C C C C C T T T
T T T G T G T T T G G T/G T T T T T T T T T T
C C C C/T C C C T T C C C C C C C C C C C T T
Additional SNPs e e e 637 e e e 542 e e e 637 e e e e e e e e 400 e
(A/G)
(G/A); 728 (T/A)
(A/A/G)
(T/G); 674 (G/C); 679 (C/A)
a RdRp: position 888 leads to (thr/thr); 1242 (ala/ala); 1972 & 1973 (ala/thr); 1973 (ala/val); 2443 (thr/ala); 2173 (met/val); 2467 (phe/val); 2583 (asp/ glu); 2598 (his/gln). b CP: position 372 leads to (val/ala); 400 (ile/met); 542 (val/ile); 637 (ser/ser); 674 (glu/gln); 679 (asn/lys); 700 (val/val); 728 (ser/thr); 888 (iso/thr). c GenBank reference HM453713 and HM453718.
countries (Table 3). The 50 UTR showed a deletion of two adenines in positions 30 and 31 in three cases (#21, 24 and 94). No other variation was detected in this part of the virus genome. In the 30 UTR, five SNPs were found in more than one sample and occurred at position 1322 (A/G), 1394 (A/C), 1449 (C/T), 1464 (G/A) and 1504 (A/G). Three SNPs (1461, 1475, 1482) were found only in single samples. The highest number of SNPs in comparison to the reference genome of the French sample #24 occurred in two samples of Argentina (#1) and Bulgaria (#8). Two pairs samples (from Morocco and Turkey; from Canada and India) were identical to each other. To test the impact of these nucleotide exchanges on the predicted secondary structure of the RNA, a minimum free energy model (Zucker & Stiegler 1981) was employed to calculate the theoretical folding in the UTRs. In case of the 50 UTR the deletion had no effect on the loop structure (loop at position 25e32). Structural analyses of the 30 UTR showed that it probably contains five prominent loops beginning at position 1338. The four SNPs in these loops (1394; 1449; 1464; 1504) apparently had no impact on the secondary structure (Fig 2B). The SNP in position 1322 is predicted to be involved in an unpaired loop and not influencing the secondary structure of the RNA.
Discussion Since its first observation in a sunflower downy mildew isolate from North America (Gulya et al. 1990), PhV could be traced by scanning electronic microscopic analysis in several samples
of the Plasmopara halstedii from different countries and of different race pathotype (Heller-Dohmen et al. 2008). However, a broad screening of the geographic distribution of the virus and on the time frame of its presence had not been attempted yet. The access to P. halstedii samples from three collections in North America, Hungary, and Germany now provided the possibility to search for PhV sequences in samples from all four continents on which sunflower downy mildew has been reported to occur. Moreover, this was a chance to see whether the virus contemporarily spread with its host or has developed more recently in different populations. In contrast to the previous study of Heller-Dohmen et al. (2008) which used TEM detection identify virus particles, the current study was PCR-based and aimed to amplify sequence parts of the two viral RNA strands encoding for the virus polymerase and the CP. To avoid false negative results in because of RNA sequence variation or RNA degradation, we used several primer sets for different sites of both ORFs. These primers were designed on the base of recently published genome sequences of five PhV samples (Heller-Dohmen et al. 2011) and aimed to amplify relatively short fragments, expected to occur particularly in old samples of P. halstedii (Table 4). Surprisingly, all samples dating older than 1988 were tested positive for PhV, no matter, whether the material had been stored for decades at room temperature on infected herbarized sunflower leaves or as sporangia in the freezer. On the one hand, this probably is due to the large amount of virions in the cytoplasm of the oomycete as was shown by Heller-Dohmen et al. (2008). On the other hand it shows that the RNA of PhV is very stable
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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W. Grasse, O. Spring
Fig 1 e Global distribution of PhV. Number of PhV containing versus total number of Plasmopara halstedii samples are indicated below the country of sample origin. The map template is provided by http://www.freeworldmaps.net/pdf/maps.html.
and well protected from degradation perhaps by mechanisms such as RdRp proofreading or endcapping as recently summarized by Dickson & Wilusz (2011) and Barr & Fearns (2010). The successful extraction of RNA from herbarized sunflower leaves infected with PhV parallels similar reports from plant virus studies performed on herbarium specimens up to the age of 100e150 y (Fraile et al. 1997; Malmstrom et al. 2007). Recently RNA sequencing of the barley stripe mosaic virus was achieved even from archeological samples 750 y old (Smith Q6 et al. 2014). In comparison to TEM studies, the PCR-based search for PhV appears to be much faster, cheaper, and more sensitive, but the amplification of viral RNA may not be fully accepted as proof for the presence of virus, because in other oomycetes viral RNA has been found although no virions were detected (Tooley et al. 1998). This has not yet been reported for P.
halstedii. Moreover, 12 out of our 116 positive tested samples were previously shown to contain virus particles by means of TEM (see highlighted samples in Table 1). Two samples which had been tested negative in the TEM study of HellerDohmen et al. (2008) also gave negative results in PCR with all primer sets. The presence of PhV in more than 90 % of the samples confirmed earlier results of Heller-Dohmen et al. (2008) who also reported a high rate of PhV infected P. halstedii isolates. A comparison with other oomycete viruses such as SMV A, SMV B (Yokoi et al. 1999, 2003) or Phytophthora infestans RNA virus (Cai et al. 2009, 2012, 2013) is not possible because of the lack of published data. However, a recent study on different partitivirus strains in Heterobasidion species showed a much lower degree of virus infection, reaching only about 5 % (Vainio et al. 2011). A reason for the high rate of infection in P. halstedii
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Table 3 e SNPs in the untranslated regions of RNA2. Sample #24 (France 1999) served as reference genome. Sample
24 94 21 95 113 82 13 93 1 3 8 2
Country
Francea Italy France Morocco Turkey Hungary Canada India Argentina Argentina Bulgaria Argentina
Date
1999 1998 1989 1997a 1997a 1988 1996a 1995 1999 2000b 1990 2000a
CP UTR 30 - -/AA
1322 G/A
1394 A/C
1449 T/C
1461 G/A
1464 G/A
1475 T/G
1482 A/C
1504 G/A
---AA AA AA AA AA AA AA AA AA
G G G G G G G G A A A A
A A C C C A A A A A A A
T C C C C T C C C C C T
G G G G G G G G A G G G
G G G G G G G G A G A G
T T T T T T T T G T T T
A A A A A A A A A A C A
G G A A A A G G G G G G
a GenBank reference HM453718.
Fig 2 e Scheme of the two RNA strands of PhV and positioning of constant SNPs. (A) RNA 1 and 2 of PhV with highlighted SNPs. Numbers indicate positions of SNP, the nucleotide exchanged and the effect on amino acid exchange. Sequenced parts are marked by grey bars. (B) Theoretical secondary structure of 30 UTR of RNA 2 according to minimum free energy modelling. Numbers indicate positions of SNPs e : gap position; A: adenine; C: cytosine; G: guanidine; U: uracil; Ala: alanine; Iso: isoleucine; Thr: threonine; Val: valine. Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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W. Grasse, O. Spring
Table 4 e Primers designed for PhV and used for screening and sequencing. Primer 2f3a,b PHV_WG_R1a PHV_WG_F1a PHV_WG_R2a PhV_Coat_UTR5_F1 PhV_Coat_UTR3_R4 PHV_pol_F1a PHV_pol_R1a PHV_pol_F2a PHV_pol_R2a PHV_pol_F5 PHV_pol_R5 PHV_pol_R7 PhV_RdRp_UTR5_F2 PhV_RdRp_UTR3_R2
Sequence 50 e30
Target
50
30
Sizec
CTGGGTAGTGGAGACTACACA TCCCTGCGATTCCACTGAGC TCAAGGGATCGATCGTATTCACA GGGTTCTCTTTGGGATCACATTC TAAACAGCCCCGACGCAG TCGCCTATGCGGGTCTCC CATATGGCCTCCGGAAGACAATC TAACACTGATTTTTCCCGCTTTGA TCCGACCTGAATACACGAATGA GGTCCATAAAGCCGGTTCAAA TTCCCGGTTTAAAATCGTGAG TCAGGTCGGAAAGCCAATAA GGGCTTCCTGCGGTTTG ATCACGCAGAGTACACTATG TGGATACTTAGGACATTGGC
CP CP CP CP CP CP RdRp RdRp RdRp RdRp RdRp RdRp RdRp RdRp RdRp
222 428 355 657 1 1510 1674 1946 1754 2124 822 1744 2738 1 2773
242 447 377 679 18 1528 1697 1968 1775 2144 842 1763 2754 20 2793
225 324
294 390
a Primers where used for the screening of PhV. b Heller-Dohmen et al. 2011. c Size of the PCR fragments in the screening experiments.
might be the fact that PhV is not a cryptic virus like most other mycoviruses (Buck 1998) which spread only intracellularly. In contrary, PhV easily infects its host from the environment as was recently shown by virus transmission experiments in which virus-free strains of P. halstedii became PhV infected when zoospores of the oomycete during the inoculation of sunflower seedlings got in contact with virus (Grasse et al. 2013). Therefore, the coexistence of virus-free and virus-containing populations of P. halstedii in the same field is not very likely, and under laboratory conditions this is only achievable by applying strict quarantine measures when culturing different isolates at a time. Moreover, we never observed the spontaneous loss of PhV in a P. halstedii isolate cultured over more than 20 y in our lab (data not shown). Apparently virus-free samples of P. halstedii were found in the US, Argentina and in the European countries of France, Germany, and Switzerland. These samples dated from 1988 to 2011 thus giving no hints for any geographic or temporal correlation of the spreading of PhV from a specific area or source. Although our sample material did not reach back to the period of introduction of P. halstedii into Europe in the 1940s (Sackston 1981) we assume that PhV may have existed already in the very early years of sunflower downy mildew infections at the beginning of the last century (Viranyi & Spring 2011). It could be of future interest to test the type specimens of Eupatorium purpureum on which Farlow described P. halstedii (Farlow 1883) in order to see whether the occurrence of PhV is linked to the host-jump of the oomycete from wild species to cultivated sunflower. At least so far, the virus was not detected in any P. halstedii isolate from wild Asteraceae (Grasse et al. 2013). The sequence analysis provided here from 22 amplified PhV sequences from P. halstedii samples from 13 different countries is the first study on global whole genome variation of a mycovirus. For that reason, comparison with other mycoviruses is not possible. The high conservation of the viral genome observed from partial sequences of PhV recently
reported by Heller-Dohmen et al. (2011) was confirmed by our results and several SNP positions (e.g. RdRp 1242 and 1972; CP 372) extractable from GenBank coincided with SNPs found in our study. Moreover, extended information was provided now for the full coding parts and for samples of a much broader range of origin and age. Nevertheless, the observed genetic diversity in the two ORFs remained extremely low (only 0.003 % and 0.008 % of the nucleotides of the RdRp and CP, respectively, were exchanged within the pool of all samples). Obviously, both strands are highly conserved which implies that mutations other than the ones observed would have negative effects on the functionality of the RdRp and CP. Studies from plant viruses also showed low genetic diversity (0.01e0.14) among different strains of the same virus from different regions (Garcıa-Arenal et al. 2001), but even the nucleotide diversity of 0.014e0.058 reported for Cucumber mosaic virus (Nouri et al. 2014) was still one dimension higher than in PhV. The low genetic diversity among plant viruses has been attributed to the lack of an immune system in plants and the missing selection pressure (Garcıa-Arenal et al. 2001). This argument may account for mycoviruses as well. In addition it appears possible that the RdRp of PhV has a proofreading function which also could contribute to conserve the sequence. Proofreading RdRps were reported to have an error rate of ca. 10-3 to 10-4 (Barr & Fearns 2010). This could explain the range of genetic diversity observed for PhV in this study. Unexpectedly the low variability of PhV expanded into the URL regions as well, although these parts are not translated into amino acids and therefore changes would not be expressed in the phenotype where they could cause malfunction. In total 10 out of 398 nt. (0.025 %) of the RNA 2 were affected by deletions or base exchanges. This is a ca. three times higher rate of diversity than in the ORF part of this strand. The assumption that alterations in these noncoding parts may have negative effects on regulatory processes of the virus reproduction could not be confirmed by our
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modelling experiments which did not predict any significant changes in the secondary structure of the RNA in samples with SNPs.
Acknowledgements We gratefully acknowledge the support by T. J. Gulya (USDA, € do € llo €, Fargo, USA) and F. Viranyi (Szent Istvan University, Go Hungary) in providing samples of P. halstedii isolates. The project was funded by the Deutsche Forschungsgemeinschaft (SP292/21-2).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2014.12.004.
references
Barr JN, Fearns R, 2010. How RNA viruses maintain their genome integrity. Journal of General Virology 91: 1373e1387. Buck K, 1998. Molecular variability of viruses of fungi. In: Bridge PD, Couteaudier Y, Clackson JM (eds), Molecular Variability of Fungal Pathogens. CAB International, Wallingford, UK, pp. 53e72. Cai G, Myers K, Hillman BI, Fry WE, 2009. A novel virus of the late blight pathogen, Phytophthora infestans, with two RNA segments and a supergroup 1 RNA-dependent RNA polymerase. Virology 392: 52e61. Cai G, Myers K, Fry WE, Hillman BI, 2012. A member of the virus family Narnaviridae from the plant pathogenic oomycete Phytophthora infestans. Archives of Virology 157: 165e169. Cai G, Krychiw JF, Myers K, Fry WE, Hillman BI, 2013. A new virus from the plant pathogenic oomycete Phytophthora infestans with an 8 kb ds RNA genome: the sixth member of a proposed new virus genus. Virology 435: 341e349. Dickson AM, Wilusz J, 2011. Strategies for viral RNA stability: live long and prosper. Trends in Genetics 27: 286e293. Farlow WG, 1883. Note on some species in the third and eleventh centuries of Ellis’s North American Fungi. Proceedings of the American Academy of Arts and Science 18: 71e73. Fraile A, Escriu F, Aranda MA, Malpica JM, Gibbs AJ, GarcıaArenal F, 1997. A century of tobamovirus evolution in an Australian population of Nicotiana glauca. Journal of Virology 71: 8316e8320. Garcıa-Arenal F, Fraile A, Malpica JM, 2001. Variability and genetic structure of plant virus populations. Annual Review of Phytopathology 39: 157e186. Grasse W, Zipper R, Totska M, Spring O, 2013. Plasmopara halstedii virus causes hypovirulence in Plasmopara halstedii, the downy mildew pathogen of the sunflower. Fungal Genetics and Biology 57: 42e47.
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Gulya TJ, Freeman TP, Mayhew DE, 1990. Virus-like particles in Plasmopara halstedii, Sunflower Downy Mildew. Phytopathology 80: 1032. Gulya T, Rashid KY, Masirevic SM, 1997. Sunflower diseases. In: Schneider AA (ed.), Sunflower Technology and Production. American Society of Agronomy, Madison, pp. 263e380. Gulya TJ, 2007. Distribution of Plasmopara halstedii races from sunflower around the world. In: Lebeda A, SpencerPhillips PTN (eds), Advances in Downy Mildew Research, vol. 3. Palacky University and JOLA Publishers, Olomouc, CZ, pp. 121e134. Han K, Byun Y, 2003. PseudoViewer2: visualization of RNA pseudoknots of any type. Nucleic Acids Research 31: 3432e3440. € pfert JC, Hammerschmidt R, Spring O, 2008. Heller-Dohmen M, Go Different pathotypes of the sunflower downy mildew pathogen Plasmopara halstedii all contain isometric virions. Molecular Plant Pathology 9: 777e786. € pfert JC, Pfannenstiel J, Spring O, 2011. The Heller-Dohmen M, Go nucleotide sequence and genome organization of Plasmopara halstedii virus. Virology Journal 8: 123e131. Hofacker IL, Fontana W, Stadler PF, Bonhoeffer S, Tacker M, Schuster P, 1994. Fast folding and comparison of RNA sec€ r Chemie 125: 167e188. ondary structures. Monatshefte fu Malmstrom CM, Shu R, Linton EW, Newton LA, Cook MA, 2007. Barley yellow dwarf viruses (BYDVs) preserved in herbarium specimens illuminate historical disease ecology of invasive and native grasses. Journal of Ecology 95: 1153e1166. Nouri S, Arevalo R, Falk BW, Groves RL, 2014. Genetic structure and molecular variability of cucumber mosaic virus isolates in the United States. PLoS ONE 9: e96582. Sackston WE, 1981. Downy mildew of sunflower. In: Spencer DM (ed.), The downy mildews. Academic, pp. 545e575. Smith O, Clapham A, Rose P, Liu Y, Wang J, Allaby RG, 2014. A complete ancient RNA genome: identification, reconstruction and evolutionary history of archeological barley stripe mosaic virus. Scientific Reports 4: 4003. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731e2739. Tooley PW, Hewings AD, Falkenstein KF, 1998. Detection of double-stranded RNA in Phytophthora infestans. Phytopathology 79: 470e474. €a € J, Dai Y-C, Hansen E, Korhonen K, Hantula J, Vainio EJ, Hakanpa 2011. Species of Heterobasidion host a diverse pool of partitiviruses with global distribution and interspecies transmission. Fungal Biology 115: 1234e1243. Viranyi F, Spring O, 2011. Advances in sunflower downy mildew research. European Journal of Plant Pathology 129: 207e220. Yokoi T, Takemoto Y, Suzuki M, Yamashita S, Hibi T, 1999. The nucleotide sequence and genome organization of Sclerophthora macrospora Virus B. Virology 264: 344e349. Yokoi T, Yamashita S, Hibi T, 2003. The nucleotide sequence and genome organization of Sclerophthora macrospora Virus A. Virology 311: 394e399. Zuker M, Stiegler P, 1981. Optimal computer folding of large RNA sequences using thermodynamic and auxiliary information. Nucleic Acids Research 9: 133e148.
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Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world Q7
Wolfgang GRASSE, Otmar SPRING* Institute of Botany, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany
Q3
Q4
article info
abstract
Article history:
Plasmopara halstedii virus (PhV) is a ss(þ)RNA virus that exclusively occurs in the sunflower
Received 16 September 2014
downy mildew pathogen P. halstedii, a biotrophic oomycete of severe economic impact. The
Received in revised form
origin of the virus and its genomic variability in host populations from different geographic
28 November 2014
regions is unknown.
Accepted 8 December 2014
Sporangia of 128 samples of P. halstedii from five continents, collected over the past 40 y, were screened for presence of PhV using PCR-based molecular tools. PhV RNA was found in over 90 % of all P. halstedii isolates with no correlation to any specific
Q5
Keywords:
geographic origin or pathotype of its host. Sequence analyses of the open reading frames
Genome diversity
(ORFs) were performed to show the genetic diversity of the virus. Only 18 single nucleotide
Mycovirus
polymorphisms (SNPs) were found in the 3873 sequenced nucleotides of the two ORFs and
Oomycetes
these SNPs had no recognizable effect on the encoded proteins. In the 398 nucleotides of
ss(þ)RNA
the untranslated regions (UTRs) of the RNA 2 strand eight additional SNPs and one short
Sunflower downy mildew
deletion was found. Modelling experiments revealed no effects of these variations on the secondary structure of the RNA. The results showed the presence of PhV in P. halstedii isolates of global origin and the existence of the virus since more than 40 y. The virus genome revealed a surprisingly low variation in both coding and noncoding parts and none of the differences was correlated with the host pathotype or geographic populations of the oomycete. ª 2014 Published by Elsevier Ltd on behalf of The British Mycological Society.
Introduction Plasmopara halstedii virus (PhV) is one of the rare characterized viruses in obligate biotrophic Oomycetes (Yokoi et al. 1999, 2003; Heller-Dohmen et al. 2011). PhV is a ss(þ)RNA virus with two RNA segments encoding for the RNA depending RNA polymerase (RNA strand 1, ORF 2745 nucleotides) and the coat protein (RNA strand 2, ORF 1128 nucleotides), respectively. Its virions are isometric, measure approximately 37 nm
in diameter, and were found in all developmental stages of its exclusive host P. halstedii, the downy mildew pathogen of sunflower (Heller-Dohmen et al. 2008, 2011). PhV occurs in the cytoplasm of hyphae, haustoria, and sporangia of its host and may reach very high numbers as was shown in transmission electron microscopy studies by Heller-Dohmen et al. (2008). A hypovirulence effect of virus infection on the aggressiveness (intensity and speed of sporulation), but not on the pathogenicity (infectivity to differentiating host genotypes) of the
* Corresponding author. Tel.: þ49 711 459 23811; fax: þ49 711 459 23355. E-mail address: [email protected] (O. Spring). http://dx.doi.org/10.1016/j.funbio.2014.12.004 1878-6146/ª 2014 Published by Elsevier Ltd on behalf of The British Mycological Society.
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oomycete was recently shown by infection experiments with two genetically homogenous strains with and without virus (Grasse et al. 2013). While P. halstedii was reported to occur in all major areas of sunflower cultivation except for Australia (Gulya et al. 1997; Gulya 2007; Viranyi & Spring 2011), the distribution of PhV in samples of P. halstedii from different geographical origins has been investigated only in a very limited number of samples (Heller-Dohmen et al. 2008, 2011). In particular the genomic variation of PvH from different origins has not been investigated in detail to date. This is similar to most known mycoviruses and is most likely due to the lack of suitable sample material. Only recently, a study was published on the occurrence of partitiviruses in seven Heterobasidion species where it was found that only 5 % of the fungi were virusinfected. Three different strains of the HetRV virus were found in ten samples, but no detail on the sequence variation in the virus genome was provided (Vainio et al. 2011). Our intensive collecting of P. halstedii isolates in Central Europe and South America as well as access to sample material from East Europe (F. Viranyi, Szent Istvan University, € do € llo € , Hungary), Africa, Asia, and North America (T.J. Gulya, Go USDA Fargo, U.S.A.) enabled us to screen for the occurrence of PhV in samples from the past 40 y. We here report on a PCRbased method for the detection of the ss(þ)RNA virus in sporangia of the oomycete. Moreover, we investigated the genetic diversity of PhV by sequence comparison of the open reading frames (ORFs) encoding for the capsid protein (CP) and the RNA depending RNA polymerase (RdRp) and by comparing the untranslated regions (UTRs) of the genome.
Methods Origin of the used Plasmopara halstedii isolates In this study, 128 Plasmopara halstedii isolates collected within the past 40 y from 17 countries on five continents were screened for the presence of PhV. The sample material either consisted of freshly harvested sporangia, sporangia which had been stored for up to ca. 40 y in the freezer, or downy mildew infected sunflower leaves with visible sporulation from herbarized specimens. The origin, year collected, condition, and provider of samples are listed in Table 1.
Nucleic acid extraction The total RNA was extracted using the BioRAD Aurum RNA Extraction Kit (Bio-Rad Laboratories, Hercules, US-CA). In case of herbarized samples, polyclearAT (1 mg/10 mg sample) was added to bind residual of phenolic substances. The RNA was transcribed into cDNA according to the manufacturer’s protocol using RevertAid reverse Transcriptase (Thermo Fisher Scientific; US-MA) and a random hexamer primer.
PCR-based virus screening The screening of the PhV samples was performed by PCR using four primer pairs specific for short sequences of both strands of the virus genome (Table 2). The primers
W. Grasse, O. Spring
PHV_pol_F1/R1 and PHV_pol_F2/R2 were used to amplify a 294 and a 390 bp fragment of the RdRp gene, respectively. The primers 2f3/PHV_WG_R1 and PHV_WG_F1/R2 were employed to amplify a 225 bp and a 324 bp fragment of the coat protein gene. PCRs were performed using Genaxxon RedTaq Mix (Genaxxon, Ulm, Germany). PCR amplifications were performed on a peqSTAR thermocycler (Peqlab, Erlangen, Germany) using the following cycling protocol: initial denaturation at 94 C for 3 min followed by 40 cycles with denaturation at 94 C for 20 s, annealing at 60 C for 20 s and elongation at 72 C for 20 s; the final extension was carried out at 72 C for 5 min. The PCR products were analysed on 2 % Agarose gels (1 TBE buffer, ethidium bromide staining). Samples were considered to be virus-free when they had been tested negative in two independent experiments.
Amplification and sequencing of cDNA PCR of cDNA amplifications were performed on a peqSTAR thermocycler (Peqlab, Erlangen, Germany) using the Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Karlsruhe) and the following cycling protocol: initial denaturation at 98 C for 3 min followed by 40 cycles with denaturation at 98 C for 20 s, annealing at 55e60 C for 20 s and elongation at 72 C for 120 s; the final extension was carried out at 72 C for 10 min. The PCR products were analysed on 1.5 % Agarose gels (1 TBE buffer, ethidium bromide staining). PCR products of the ORFs and the UTRs generated with specific primers (for used primers see Table 2) were sequenced commercially (Macrogen, Amsterdam, Netherlands). For sequence comparison the sequences of PhV coat HM453718 and RdRp HM453713 of sample #24 (a virus isolate from France 1999) published by Heller-Dohmen et al. (2011) were used as reference. The alignment was done with clustalW algorithm from the software BioEdit (Ibis Bioscience, USA). The mean distance was estimated with the program MEGA5 (Tamura et al. 2011).
Modelling of 30 UTR structure The secondary RNA structure of the 30 UTR was performed with the minimal free energy algorithm of the Vienna RNA webserver (Zucker & Stiegler 1981; Hofacker et al. 1994). For the visualization of the secondary RNA structure the software PseudoViewer 2.5 was used (Han & Byun 2003).
Results Occurrence of PhV in Plasmopara halstedii samples of different continents PhV viral RNA was successfully extracted and translated into cDNA from Plasmopara halstedii sporangium samples which were collected up to 40 y ago. The screening with four specific primer pairs (Table 1) for short sequences of both RNA strands showed the presence of PhV in almost all cases, no matter whether freeze-stored sporangia, freshly harvested sporangia or sunflower leaves with visible sporulation from old herbarized plant specimens were used. Out of 128 samples of different
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
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3
Q1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Table 1 e Origin and collection dates of P. halstedii samples used for the PCR-based PhV screening. Samples used for sequencing are highlighted in dark grey; samples analysed by transmission electron microscopy in the previous study of Heller-Dohmen et al. (2008) are marked in light grey. Sample #
Provided
a
b
c
44
OS
Germany
2003d
hl
+
45
OS
Germany
2003e
hl
+
46
OS
Germany
2004a
hl
+
47
OS
Germany
2004b
hl
+
48
OS
Germany
2004c
hl
+
Country
Date
Source
PhV
49
OS
Germany
2004d
hl
+
1
TG
Argentina
1999
fs
+
50
OS
Germany
2004e
hl
+
2
TG
Argentina
2000a
fs
+
51
OS
Germany
2005a
hl
+
3
TG
Argentina
2000b
fs
+
52
OS
Germany
2005b
hl
+
4
OS
Argentina
2003a
hl
+
53
OS
Germany
2005c
hl
+
5
OS
Argentina
2003b
hl
-
54
OS
Germany
2005d
hl
+
6
OS
Austria
1999a
hl
+
55
OS
Germany
2005e
hl
+
7
OS
Austria
1999b
hl
+
56
OS
Germany
2008a
hl
+
8
TG
Bulgaria
1990
fs
+
57
OS
Germany
2008b
hl
+
9
TG
Bulgaria
1999
fs
+
58
OS
Germany
2008c
hl
+
10
TG
Canada
1991
fs
+
59
OS
Germany
2010a
s
-
11
TG
Canada
1992
fs
+
60
OS
Germany
2010b
s
+
12
TG
Canada
1995
fs
+
61
OS
Germany
2010c
s
-
13
TG
Canada
1996a
fs
+
62
OS
Germany
2010d
s
+
14
TG
Canada
1996b
fs
+
63
OS
Germany
2010e
s
-
15
TG
Canada
1998
fs
+
64
OS
Germany
2011a
s
+
16
OS
Canada
2001
hl
+
65
OS
Germany
2011b
s
-
17
OS
Canada
2002
hl
-
66
OS
Germany
2011c
s
-
18
FV
China
1987
hl
+
67
OS
Germany
2012a
s
+
19
TG
China
1992
fs
+
68
OS
Germany
2012b
s
+
20
TG
France
1988
fs
-
69
OS
Germany
2012c
s
+
21
TG
France
1989
fs
+
70
OS
Germany
2012d
s
+
22
TG
France
1991
fs
+
71
OS
Germany
2012e
s
-
23
TG
France
1998
fs
+
72
OS
Germany
2012f
s
+
24
OS
France
1999
hl
+
73
OS
Germany
2012g
s
+
25
TG
France
2000a
fs
+
74
OS
Germany
2012h
s
+
26
OS
France
2000b
hl
+
75
OS
Germany
2012i
s
+
27
OS
France
2000c
hl
+
76
OS
Germany
2012j
s
+
28
OS
France
2000d
hl
+
77
FV
Hungary
1972
hl
+
29
OS
France
2000e
hl
+
78
FV
Hungary
1974
hl
+
30
TG
Germany
1992
fs
+
79
FV
Hungary
1976
hl
+
31
OS
Germany
1993
hl
+
80
FV
Hungary
1984a
hl
+
32
OS
Germany
1996
hl
+
81
FV
Hungary
1984b
hl
+
33
OS
Germany
1997a
hl
+
82
TG
Hungary
1988
fs
+
34
OS
Germany
1997b
hl
+
83
FV
Hungary
1989
hl
+
35
OS
Germany
1998a
hl
+
84
TG
Hungary
1990a
fs
+
36
OS
Germany
1998b
hl
+
85
TG
Hungary
1990b
fs
+
37
OS
Germany
2000
hl
+
86
TG
Hungary
1991
fs
+
38
OS
Germany
2001a
hl
+
87
TG
Hungary
1992
fs
+
39
OS
Germany
2001b
hl
+
88
TG
Hungary
1993
fs
+
40
OS
Germany
2001c
hl
-
89
TG
Hungary
1995a
fs
+
41
OS
Germany
2003a
hl
+
90
FV
Hungary
1995b
hl
+
42
OS
Germany
2003b
hl
+
43
OS
Germany
2003c
hl
+
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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W. Grasse, O. Spring
91
OS
Hungary
2001a
hl
+
92
OS
Hungary
2001b
hl
+
93
TG
India
1995
fs
+
94
TG
Italy
1998
fs
+
95
TG
Morocco
1997a
fs
+
96
TG
Morocco
1997b
fs
+
97
TG
Morocco
1997c
fs
+
98
TG
Morocco
1998
fs
+
99
OS
Slovakia
1999
hl
+
100
TG
South Africa
1996a
fs
+
101
TG
South Africa
1996b
fs
+
102
TG
South Africa
1996c
fs
+
103
TG
South Africa
1997a
fs
+
104
TG
South Africa
1997b
fs
+
105
TG
Spain
1986
fs
+
106
TG
Spain
1994
fs
+
107
TG
Spain
1997
fs
+
108
TG
Spain
1998
fs
+
109
TG
Spain
1999
fs
+
110
OS
Switzerland
2011
hl
-
111
TG
Turkey
1996a
fs
+
112
TG
Turkey
1996b
fs
+
113
TG
Turkey
1997a
fs
+
114
TG
Turkey
1997b
fs
+
115
TG
Turkey
1997c
fs
+
116
TG
USA
1985a
fs
+
117
TG
USA
1985b
fs
+
118
TG
USA
1988
fs
+
119
TG
USA
1989
fs
+
120
TG
USA
1991a
fs
+
121
TG
USA
1991b
fs
+
122
TG
USA
1992
fs
+
123
OS
USA
1993
hl
+
124
TG
USA
1994
fs
+
125
OS
USA
1997
hl
+
126
TG
USA
1998
fs
+
127
TG
USA
1999
fs
+
128
OS
USA
2007
hl
-
age and origin, 117 tested positive which indicates that more than 90 % of the P. halstedii isolates harboured the virus genome (Fig 1; Table 2). In all positive samples, amplification products of both RNA strands were found. No correlation was found between the geographic origin of samples, and presence or absence of PhV sequences. Virus-free samples were found among the samples from North America (2 out of 21), South America (1 out of 5), and Europe (8 out of 91), whereas samples from Africa (9) and Asia (3) all contained PhV sequences. Negative PCR results occurred in samples from fresh sporangia as well as from herbarized specimens. With respect to the seemingly higher rate of virus-free samples in Germany (6 out of 45) and Switzerland (1 out of 1) it is noteworthy that the majority of collections in these countries derived from small fields of cutting sunflowers where mostly sunflower seeds with unknown breeding history were cultivated and no chemical plant protection against downy mildew was employed. The occurrence of PhV was not correlated with the sampling date of the P. halstedii isolate. The oldest sample containing PhV sequences was
collected in 1972 in Hungary, while the oldest virus-free sample was from France collected in 1988.
Sequence variation in the two ORFs Short PCR fragments of the two coding regions for the RdRp and CP of PhV could readily be amplified for all isolates. In contrast, longer amplicons were sometimes difficult to obtain, possibly because the RNA might have been partly degraded over the time of storage. Sequencing of the complete ORFs (2745 nucleotides for RdRp; 1128 nucleotides for CP) was achieved for 22 samples from 13 countries. Alignments of the 3873 bp complete ORFs showed five positions in which single nucleotide polymorphisms (SNPs) consistently occurred. In addition, 13 SNPs only occurred in sequences of one or two samples (position numbers as given in Table 2; for base identity see Additional File 1, 2). Compared with reference genome of PhV reported for sample #24 (GenBank PhV coat HM453718 and RdRp HM453713) the five consistent positions were nt. 1242 (A/G exchange) and nt. 2443 (A/G) in case of the RdRp (RNA1), and position nt. 372 (C/ T), nt. 700 (T/G) and nt. 888 (C/T) in case of the coat protein (RNA2), respectively (Table 2). These five SNPs lead to amino acid exchanges in only three cases, because SNP RNA1 1242 and SNP RNA2 700 were silent mutations. SNP RNA1 2443 accounted for an alanine/threonine exchange and occurred in 18 of the 22 samples. The SNPs RNA2 372 and 888 caused an alanine/valine and threonine/isoleucine exchange in the coat protein, respectively. The former was found in 15, the latter occurred in five of the 22 samples. The 13 additional SNPs led to 11 amino acid changes in the RdRp (nt. 1972 alanine/threonine, nt. 1973 alanine/valine, nt. 2173 methionine/valine, nt. 2467 phenylalanine/valine, nt. 2583 aspartate/glutamate and nt. 2598 histidine/glutamine) and the coat protein (nt. 400 isoleucine/ methionine, nt. 542 valine/isoleucine, nt. 674 glutamate/glutamine, nt. 679 asparagine/lysine, and nt. 728 serine/threonine). The highest accumulation of SNPs (8) compared to the reference sequences appeared in an Argentinian sample from 1999 (#1). None of the sequenced samples was completely identical with the reference. The lowest number of SNPs (1) in the ORFs was found in two samples from Canada (#13, 1996a) and Germany (#51, 2005a). In no case we observed a correlation of SNPs with the origin or age of the samples and no coincidence between nucleotide exchanges in the RdRp versus CP became visible. A group of seven samples (indicated by sidebar in Table 2) showed identical ORFs, but their origin from three continents and sampling dates over a period of 12 y does not imply an internal relationship. In four samples (Bulgaria 1999; USA 1999; Argentina 2000b; Germany 2003) mixed populations of two different PhV genotypes was detected. Within the complete ORFs the genetic diversity of the 22 samples was extremely low with a mean of distance of 0.001 for the RdRp and 0.002 for the CP.
Sequence variation in the UTRs The presumable amplicons of the UTRs of the RNA1 strand (RdRp) were too short (18 nt; 30 nt) for sequence comparison outside of the primer binding sites. Therefore, we focused on the UTRs of RNA2 (CP) which consisted of 166 nt and 234 nt for the 50 and 30 end, respectively. Complete sequences of these regions were obtained from 12 samples out of 9
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
5
Table 2 e Position of SNPs in ORFs of PhV isolates. Sample #24 (France 1999) served as reference genome. SNPs found in only one or two samples are indicated by the nt. position. Samples identical in sequence are marked by side bar. Sample
Country
RdRpa
Date
1242 A/G 2443 G/A 24 43 51 9 58 89 13 2 3 11 93 127 21 94 95 100 105 122 124 113 1 8
c
France Germany Germany Bulgaria Germany Hungary Canada Argentina Argentina Canada India USA France Italy Morocco S. Africa Spain USA USA Turkey Argentina Bulgaria
1999 2003c 2005a 1999 2008c 1995a 1996a 2000a 2000b 1992 1995 1999 1989 1998 1997a 1996 1986 1992 1994 1997a 1999 1990
A A/G A A A A A A A/G G G G G G G G G G G G G G
G G G G A A A A A A A A A A A A A A A A A A
CPb
Additional SNPs
372 T/C
700 T/G
888 C/T
e e e 2598 (T/A) e e e 1972 (G/A); 1973 (C/T) 2467 (T/G) 888 (C/A); 2173 (A/G) e e e e e e e e e e 1973 (C/T); 2583 (C/A) e
T C C C C C T T T C C C C C C C C C C T T T
T T T G T G T T T G G T/G T T T T T T T T T T
C C C C/T C C C T T C C C C C C C C C C C T T
Additional SNPs e e e 637 e e e 542 e e e 637 e e e e e e e e 400 e
(A/G)
(G/A); 728 (T/A)
(A/A/G)
(T/G); 674 (G/C); 679 (C/A)
a RdRp: position 888 leads to (thr/thr); 1242 (ala/ala); 1972 & 1973 (ala/thr); 1973 (ala/val); 2443 (thr/ala); 2173 (met/val); 2467 (phe/val); 2583 (asp/ glu); 2598 (his/gln). b CP: position 372 leads to (val/ala); 400 (ile/met); 542 (val/ile); 637 (ser/ser); 674 (glu/gln); 679 (asn/lys); 700 (val/val); 728 (ser/thr); 888 (iso/thr). c GenBank reference HM453713 and HM453718.
countries (Table 3). The 50 UTR showed a deletion of two adenines in positions 30 and 31 in three cases (#21, 24 and 94). No other variation was detected in this part of the virus genome. In the 30 UTR, five SNPs were found in more than one sample and occurred at position 1322 (A/G), 1394 (A/C), 1449 (C/T), 1464 (G/A) and 1504 (A/G). Three SNPs (1461, 1475, 1482) were found only in single samples. The highest number of SNPs in comparison to the reference genome of the French sample #24 occurred in two samples of Argentina (#1) and Bulgaria (#8). Two pairs samples (from Morocco and Turkey; from Canada and India) were identical to each other. To test the impact of these nucleotide exchanges on the predicted secondary structure of the RNA, a minimum free energy model (Zucker & Stiegler 1981) was employed to calculate the theoretical folding in the UTRs. In case of the 50 UTR the deletion had no effect on the loop structure (loop at position 25e32). Structural analyses of the 30 UTR showed that it probably contains five prominent loops beginning at position 1338. The four SNPs in these loops (1394; 1449; 1464; 1504) apparently had no impact on the secondary structure (Fig 2B). The SNP in position 1322 is predicted to be involved in an unpaired loop and not influencing the secondary structure of the RNA.
Discussion Since its first observation in a sunflower downy mildew isolate from North America (Gulya et al. 1990), PhV could be traced by scanning electronic microscopic analysis in several samples
of the Plasmopara halstedii from different countries and of different race pathotype (Heller-Dohmen et al. 2008). However, a broad screening of the geographic distribution of the virus and on the time frame of its presence had not been attempted yet. The access to P. halstedii samples from three collections in North America, Hungary, and Germany now provided the possibility to search for PhV sequences in samples from all four continents on which sunflower downy mildew has been reported to occur. Moreover, this was a chance to see whether the virus contemporarily spread with its host or has developed more recently in different populations. In contrast to the previous study of Heller-Dohmen et al. (2008) which used TEM detection identify virus particles, the current study was PCR-based and aimed to amplify sequence parts of the two viral RNA strands encoding for the virus polymerase and the CP. To avoid false negative results in because of RNA sequence variation or RNA degradation, we used several primer sets for different sites of both ORFs. These primers were designed on the base of recently published genome sequences of five PhV samples (Heller-Dohmen et al. 2011) and aimed to amplify relatively short fragments, expected to occur particularly in old samples of P. halstedii (Table 4). Surprisingly, all samples dating older than 1988 were tested positive for PhV, no matter, whether the material had been stored for decades at room temperature on infected herbarized sunflower leaves or as sporangia in the freezer. On the one hand, this probably is due to the large amount of virions in the cytoplasm of the oomycete as was shown by Heller-Dohmen et al. (2008). On the other hand it shows that the RNA of PhV is very stable
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
W. Grasse, O. Spring
Fig 1 e Global distribution of PhV. Number of PhV containing versus total number of Plasmopara halstedii samples are indicated below the country of sample origin. The map template is provided by http://www.freeworldmaps.net/pdf/maps.html.
and well protected from degradation perhaps by mechanisms such as RdRp proofreading or endcapping as recently summarized by Dickson & Wilusz (2011) and Barr & Fearns (2010). The successful extraction of RNA from herbarized sunflower leaves infected with PhV parallels similar reports from plant virus studies performed on herbarium specimens up to the age of 100e150 y (Fraile et al. 1997; Malmstrom et al. 2007). Recently RNA sequencing of the barley stripe mosaic virus was achieved even from archeological samples 750 y old (Smith Q6 et al. 2014). In comparison to TEM studies, the PCR-based search for PhV appears to be much faster, cheaper, and more sensitive, but the amplification of viral RNA may not be fully accepted as proof for the presence of virus, because in other oomycetes viral RNA has been found although no virions were detected (Tooley et al. 1998). This has not yet been reported for P.
halstedii. Moreover, 12 out of our 116 positive tested samples were previously shown to contain virus particles by means of TEM (see highlighted samples in Table 1). Two samples which had been tested negative in the TEM study of HellerDohmen et al. (2008) also gave negative results in PCR with all primer sets. The presence of PhV in more than 90 % of the samples confirmed earlier results of Heller-Dohmen et al. (2008) who also reported a high rate of PhV infected P. halstedii isolates. A comparison with other oomycete viruses such as SMV A, SMV B (Yokoi et al. 1999, 2003) or Phytophthora infestans RNA virus (Cai et al. 2009, 2012, 2013) is not possible because of the lack of published data. However, a recent study on different partitivirus strains in Heterobasidion species showed a much lower degree of virus infection, reaching only about 5 % (Vainio et al. 2011). A reason for the high rate of infection in P. halstedii
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
7
Table 3 e SNPs in the untranslated regions of RNA2. Sample #24 (France 1999) served as reference genome. Sample
24 94 21 95 113 82 13 93 1 3 8 2
Country
Francea Italy France Morocco Turkey Hungary Canada India Argentina Argentina Bulgaria Argentina
Date
1999 1998 1989 1997a 1997a 1988 1996a 1995 1999 2000b 1990 2000a
CP UTR 30 - -/AA
1322 G/A
1394 A/C
1449 T/C
1461 G/A
1464 G/A
1475 T/G
1482 A/C
1504 G/A
---AA AA AA AA AA AA AA AA AA
G G G G G G G G A A A A
A A C C C A A A A A A A
T C C C C T C C C C C T
G G G G G G G G A G G G
G G G G G G G G A G A G
T T T T T T T T G T T T
A A A A A A A A A A C A
G G A A A A G G G G G G
a GenBank reference HM453718.
Fig 2 e Scheme of the two RNA strands of PhV and positioning of constant SNPs. (A) RNA 1 and 2 of PhV with highlighted SNPs. Numbers indicate positions of SNP, the nucleotide exchanged and the effect on amino acid exchange. Sequenced parts are marked by grey bars. (B) Theoretical secondary structure of 30 UTR of RNA 2 according to minimum free energy modelling. Numbers indicate positions of SNPs e : gap position; A: adenine; C: cytosine; G: guanidine; U: uracil; Ala: alanine; Iso: isoleucine; Thr: threonine; Val: valine. Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
W. Grasse, O. Spring
Table 4 e Primers designed for PhV and used for screening and sequencing. Primer 2f3a,b PHV_WG_R1a PHV_WG_F1a PHV_WG_R2a PhV_Coat_UTR5_F1 PhV_Coat_UTR3_R4 PHV_pol_F1a PHV_pol_R1a PHV_pol_F2a PHV_pol_R2a PHV_pol_F5 PHV_pol_R5 PHV_pol_R7 PhV_RdRp_UTR5_F2 PhV_RdRp_UTR3_R2
Sequence 50 e30
Target
50
30
Sizec
CTGGGTAGTGGAGACTACACA TCCCTGCGATTCCACTGAGC TCAAGGGATCGATCGTATTCACA GGGTTCTCTTTGGGATCACATTC TAAACAGCCCCGACGCAG TCGCCTATGCGGGTCTCC CATATGGCCTCCGGAAGACAATC TAACACTGATTTTTCCCGCTTTGA TCCGACCTGAATACACGAATGA GGTCCATAAAGCCGGTTCAAA TTCCCGGTTTAAAATCGTGAG TCAGGTCGGAAAGCCAATAA GGGCTTCCTGCGGTTTG ATCACGCAGAGTACACTATG TGGATACTTAGGACATTGGC
CP CP CP CP CP CP RdRp RdRp RdRp RdRp RdRp RdRp RdRp RdRp RdRp
222 428 355 657 1 1510 1674 1946 1754 2124 822 1744 2738 1 2773
242 447 377 679 18 1528 1697 1968 1775 2144 842 1763 2754 20 2793
225 324
294 390
a Primers where used for the screening of PhV. b Heller-Dohmen et al. 2011. c Size of the PCR fragments in the screening experiments.
might be the fact that PhV is not a cryptic virus like most other mycoviruses (Buck 1998) which spread only intracellularly. In contrary, PhV easily infects its host from the environment as was recently shown by virus transmission experiments in which virus-free strains of P. halstedii became PhV infected when zoospores of the oomycete during the inoculation of sunflower seedlings got in contact with virus (Grasse et al. 2013). Therefore, the coexistence of virus-free and virus-containing populations of P. halstedii in the same field is not very likely, and under laboratory conditions this is only achievable by applying strict quarantine measures when culturing different isolates at a time. Moreover, we never observed the spontaneous loss of PhV in a P. halstedii isolate cultured over more than 20 y in our lab (data not shown). Apparently virus-free samples of P. halstedii were found in the US, Argentina and in the European countries of France, Germany, and Switzerland. These samples dated from 1988 to 2011 thus giving no hints for any geographic or temporal correlation of the spreading of PhV from a specific area or source. Although our sample material did not reach back to the period of introduction of P. halstedii into Europe in the 1940s (Sackston 1981) we assume that PhV may have existed already in the very early years of sunflower downy mildew infections at the beginning of the last century (Viranyi & Spring 2011). It could be of future interest to test the type specimens of Eupatorium purpureum on which Farlow described P. halstedii (Farlow 1883) in order to see whether the occurrence of PhV is linked to the host-jump of the oomycete from wild species to cultivated sunflower. At least so far, the virus was not detected in any P. halstedii isolate from wild Asteraceae (Grasse et al. 2013). The sequence analysis provided here from 22 amplified PhV sequences from P. halstedii samples from 13 different countries is the first study on global whole genome variation of a mycovirus. For that reason, comparison with other mycoviruses is not possible. The high conservation of the viral genome observed from partial sequences of PhV recently
reported by Heller-Dohmen et al. (2011) was confirmed by our results and several SNP positions (e.g. RdRp 1242 and 1972; CP 372) extractable from GenBank coincided with SNPs found in our study. Moreover, extended information was provided now for the full coding parts and for samples of a much broader range of origin and age. Nevertheless, the observed genetic diversity in the two ORFs remained extremely low (only 0.003 % and 0.008 % of the nucleotides of the RdRp and CP, respectively, were exchanged within the pool of all samples). Obviously, both strands are highly conserved which implies that mutations other than the ones observed would have negative effects on the functionality of the RdRp and CP. Studies from plant viruses also showed low genetic diversity (0.01e0.14) among different strains of the same virus from different regions (Garcıa-Arenal et al. 2001), but even the nucleotide diversity of 0.014e0.058 reported for Cucumber mosaic virus (Nouri et al. 2014) was still one dimension higher than in PhV. The low genetic diversity among plant viruses has been attributed to the lack of an immune system in plants and the missing selection pressure (Garcıa-Arenal et al. 2001). This argument may account for mycoviruses as well. In addition it appears possible that the RdRp of PhV has a proofreading function which also could contribute to conserve the sequence. Proofreading RdRps were reported to have an error rate of ca. 10-3 to 10-4 (Barr & Fearns 2010). This could explain the range of genetic diversity observed for PhV in this study. Unexpectedly the low variability of PhV expanded into the URL regions as well, although these parts are not translated into amino acids and therefore changes would not be expressed in the phenotype where they could cause malfunction. In total 10 out of 398 nt. (0.025 %) of the RNA 2 were affected by deletions or base exchanges. This is a ca. three times higher rate of diversity than in the ORF part of this strand. The assumption that alterations in these noncoding parts may have negative effects on regulatory processes of the virus reproduction could not be confirmed by our
Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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modelling experiments which did not predict any significant changes in the secondary structure of the RNA in samples with SNPs.
Acknowledgements We gratefully acknowledge the support by T. J. Gulya (USDA, € do € llo €, Fargo, USA) and F. Viranyi (Szent Istvan University, Go Hungary) in providing samples of P. halstedii isolates. The project was funded by the Deutsche Forschungsgemeinschaft (SP292/21-2).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2014.12.004.
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Please cite this article in press as: Grasse W, Spring O, Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world, Fungal Biology (2015), http://dx.doi.org/10.1016/j.funbio.2014.12.004
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