Characterization of a Novel Bipartite Double-Stranded …jvi.asm.org/content/early/2012/04/06/JVI.00292-12.full.pdf33 encompass dsRNA-1 and dsRNA-2, and three stru ctural proteins

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Characterization of a Novel Bipartite Double-Stranded RNA Mycovirus 1

Conferring Hypovirulence in the Phytopathogenic Fungus Botrytis porri 2

Mingde Wu, Fengyin Jin, Jing Zhang, Long Yang, Daohong Jiang, and Guoqing Li* 3

The State Key Laboratory of Agricultural Microbiology and the Key Laboratory of Plant 4

Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei, China. 5

* Corresponding author: Dr. Guoqing Li, 86-27-87286880, E-mail: [email protected] 6

Running title: A novel bipartite dsRNA mycovirus in Botrytis porri 7

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00292-12 JVI Accepts, published online ahead of print on 11 April 2012

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The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. Here we 21

report the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) 22

mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 23

of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 24

(5,879 bp), which share sequence identity of 62 and 95% at the 3’- and 5’-terminual regions, 25

respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were 26

detected. The protein encoded by the 3’-proximal coding region of ORF I shows sequence 27

identities of 19–23% with RNA-dependent RNA polymerases encoded by viruses in the families 28

Totiviridae, Chrysoviridae and Megabirnaviridae. However, the proteins encoded by the 29

5’-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any 30

reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade 31

distinct from those of other known RNA mycoviruses. Purified virions of ~35 nm in diameter 32

encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80 and 85 kDa, 33

respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are 34

encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified 35

virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial 36

growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite 37

dsRNA virus that possibly belongs to a new virus family. 38

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Mycoviruses (fungal viruses) have been recorded to be widespread in the major taxonomic 44

groups of filamentous fungi, yeasts and oomycetes (16, 39). Most mycoviruses have RNA 45

genomes, which are either single-stranded (ss) or double-stranded (ds), and are now classified 46

into twelve families (16). Members of four families (Chysoviridae, Partitiviridae, Reoviride and 47

Totiviridae) accommodate undivided (e.g. Totiviridae) or divided (e.g. Chysoviridae, 48

Partitiviridae and Reoviride) dsRNA genomes, which are encapsidated within capsid proteins 49

with formation of rigid virus particles. Members of six families (Alphaflexiviridae, Barnaviride, 50

Endornaviridae, Gammaflexiviridae, Hypoviridae and Narnaviridae) accommodate ssRNA 51

genomes, of which only two families (Alphaflexiviridae and Gammaflexiviridae) form virus 52

particles, whereas members of the remaining four virus families are unencapsidated and do not 53

form typical virions. Two families (Metaviridae and Pseudoviridae) accommodate RNA 54

reverse-transcribing genomes. Meanwhile, an increasing number of novel mycoviruses have been 55

reported recently (2, 9, 29, 53, 59). Chiba et al. (9) proposed to establish a new bipartite dsRNA 56

virus family, Megabirnaviridae, to accommodate Rosellinia necatrix megabirnavirus 1 57

(RnMBV1) based on characterization of the dsRNA genome of RnMBV1. Besides RNA 58

mycoviruses, Yu et al. (55) reported a geminivirus-related ssDNA mycovirus infecting 59

Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of stem rot of oilseed rape (Brassica 60

napus). 61

In many cases, mycovirus infection does not cause any visible abnormal symptoms for host 62

fungi (cryptic infections). However, infection by some mycoviruses can cause abnormal 63

symptoms in their host fungi, including reduced mycelial growth and debilitated virulence (e.g. 64

hypovirulence) (37). Successful control of chestnut blight caused by virulent strains of 65

Cryphonectria (= Endothia) parasitica with hypovirulent strains of this pathogen in Europe (1, 66


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19, 30) inspired numerous researchers to study the phenomenon of mycovirus-mediated 67

hypovirulence in plant pathogenic fungi. So far, many mycoviruses belonging to Hypoviridae, 68

Narnaviridae, Reoviridae, Megabirnaviridae and some unassigned virus taxa have been 69

identified to cause (or to be associated with) fungal hypovirulence (16, 38, 39). 70

Allium crops, including garlic (Allium sativum), garlic chives (A. tuberosum), bulb onions 71

(A. cepa) and green onions (A. fistulosum), are important vegetables grown in China, as well as in 72

many other countries. Gray mold disease caused by Botrytis spp., including B. porri, is one of the 73

most important ecological factors limiting both the yield and the quality of these Allium crops 74

(33, 57, 58). Previous studies indicated that B. porri can cause garlic clove rot (12), garlic leaf 75

blight (57) and leek leaf rot (3). Several RNA mycoviruses have been reported in B. cinerea (5, 6, 76

21, 22, 23, 50, 52), a close relative of B. porri and the causal agent of gray mold disease on 77

numerous plants. The genomes of some mycoviruses infecting B. cinerea have been 78

characterized (22, 23, 52). However, no mycoviruses infecting other species of Botrytis, 79

including B. porri, have been reported so far. 80

A strain of B. porri, designated as GarlicBc-72, was isolated from a diseased leaf of garlic 81

grown in Zhushan County, Hubei Province, China (57). We found that it grew slowly on culture 82

media and was hypovirulent to garlic leaves compared to other strains of B. porri. Two dsRNA 83

segments of approximately 6.2 and 6.5 kb in size, respectively, were detected in mycelia of strain 84

GarlicBc-72, but were not detected in virulent strains of B. porri. These results suggested that 85

strain GarlicBc-72 might be infected with an RNA mycovirus. Therefore, a study was conducted 86

to fulfill the following three objectives: (i) to establish the cause-effect relationship between 87

presence of the two dsRNA segments and hypovirulence in strain GarlicBc-72; (ii) to obtain and 88

analyze the full-length cDNA sequences generated from the two dsRNA segments; and (iii) to 89


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purify the virus particles from strain GarlicBc-72 and to characterize their nucleic acids and the 90

structural proteins at the molecular level. 91

MATERIALS AND METHODS 92

Fungal strains/isolates and biological characterization. Fifteen strains or isolates of 93

Botrytis porri were used in this study (Table 1). For simplicity, the names of strains GarlicBc-16, 94

GarlicBc-38, GarlicBc-72, OnionBc-95 and GP72SC35 of B. porri were shortened herein as 95

Bc-16, Bc-38, Bc-72, Bc-95 and SC35, respectively. Maintenance of the stock cultures and 96

establishment of working cultures of these strains of B. porri were carried out as described in our 97

previous study (51). Mycelial agar plugs (6 mm diameter) removed from the colony margin of a 98

3- to 5-day-old culture of each strain/isolate were placed on potato dextrose agar (PDA) in Petri 99

dishes (9 cm diameter), one plug per dish. The dishes were incubated at 20 ºC for determination 100

of the mycelial growth rate and for observation of the colony morphology. Pathogenicity of each 101

strain was determined by inoculating detached leaves of garlic (A. sativum cultivar Feng Suan 102

No. 1) with agar plugs of actively-growing mycelia using the procedures of Zhang et al. (57, 58). 103

Vertical transmission of hypovirulence-associated dsRNAs. Vertical transmission refers 104

to transmission of hypovirulence-associated dsRNAs from mycelia to asexual or sexual spores 105

(38). This experiment was done by determining the pathogenicity and the presence of dsRNAs in 106

single-conidium (SC) isolates from the dsRNA-containing hypovirulent strain Bc-72. For 107

obtaining conidia, strain Bc-72 was incubated at 20 ºC on PDA in Petri dishes (9 cm diameter) 108

for 17 days. Colonies showing conidial production were selected. Conidia in one of the dishes 109

were washed off by flooding the colony with ~3 ml sterile distilled water (SDW), followed by 110

scraping the surface of the colony with a sterilized glass rod. The conidial mixtures from the 111

selected dishes were pooled and filtered through four-layer cheesecloth to remove mycelial 112


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fragments. The resulting conidial suspension was diluted to 1 × 104 conidia/mL with SDW. An 113

aliquot of 100 µL conidial suspension was pipetted onto a PDA dish and the conidial suspension 114

drop was carefully spread on the PDA plate using a sterilized glass rod. Individual conidia on the 115

PDA surface were picked up using a sterilized fine glass needle under a light microscope and 116

transferred to fresh PDA plates, one conidium per plate. The plates were incubated at 20 ºC for 5 117

days and the resulting cultures were identified and those belonging to B. porri were individually 118

tested for the mycelial growth rates on PDA (20 ºC), pathogenicity on detached garlic leaves (20 119

ºC, 72 h) and the presence of the two dsRNA segments in mycelia using previously described 120

methods (51, 57, 58). The hypovirulent strain Bc-72 and the virulent strain Bc-16 of B. porri 121

were used as controls in this experiment. 122

Horizontal transmission of hypovirulence-associated dsRNAs. Horizontal transmission 123

refers to transmission hypovirulence-associated dsRNAs from hypovirulent to virulent fungal 124

strains through hyphal anastomosis (38). The experiment was done on PDA in Petri dishes using 125

the pairing culture technique (51, 52, 56). In the contact culture in each dish (9 cm diameter), the 126

dsRNA-containing hypovirulent strain Bc-72 served as the donor, whereas strains Bc-38, SC35 127

or Bc-95 served as recipients. After incubation of the contact cultures at 20 °C for 8 days, three 128

mycelial derivative isolates were obtained from three colonies of each recipient strain in the three 129

contact cultures of Bc-72/Bc-38, Bc-72/SC35 or Bc-72/Bc-95 using the method described by Wu 130

et al. (51). The isolates were designated as 72-38-1, 72-38-2 and 72-38-3 for derivatives from 131

strain Bc38; 72-35-1, 72-35-2 and 72-35-3 for derivatives from strain SC35; and 72-95-1, 132

72-95-2 and 72-95-3 for derivatives from strain Bc-95. All these derivative isolates were 133

individually tested for the mycelial growth rates on PDA (20 ºC), pathogenicity on detached 134


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garlic leaves (20 ºC, 72 h) and the presence of dsRNA in mycelia (51, 57, 58). The parental 135

strains Bc-72, Bc-38, Bc-95 and SC35 were included as controls in this experiment. 136

Transmission electron microscopy (TEM). Sub-cellular characteristics of hyphal cells of 137

strains Bc-16 (virulent) and Bc-72 (hypovirulent) were examined using TEM. A mycelial agar 138

plug of each strain was inoculated in the center of a piece of sterilized cellophane film placed on 139

PDA in a Petri dish. The culture was incubated at 20 ºC for 3 days for strain Bc-16 and for 5 days 140

for strain Bc-72. Small pieces (2 × 2 mm, length × width) of the cellophane films colonized with 141

mycelia of strains Bc-16 or Bc-72 were carefully cut off from the colony margin (~3 mm in 142

width) and the central area (~10 mm from the margin area) using a razor blade. The mycelial 143

specimens were fixed and dehydrated using conventional procedures. Ultra-thin sections (50–60 144

nm in thickness) for the mycelial specimens were cut using a diamond knife, mounted on slotted 145

and formvar-coated grids, stained with 5% aqueous lead citrate and 5% uranyl acetate, and 146

examined under TEM (Model Tecnai G2 20, FEI CompanyTM, Hillsboro, OR, USA). 147

Extraction and purification of dsRNA from mycelia. Mycelial agar plugs of each 148

strain/isolate of B. porri were inoculated in potato dextrose broth in Petri dishes (9 cm diameter), 149

1 agar plug per dish and 20 dishes per strain. The dishes were incubated at 20 ºC in the dark for 3 150

to 6 days. Mycelial mats in each dish were harvested and stored at at -20 ºC until use. Extraction 151

and purification of the dsRNAs from mycelia of each strain were performed using the procedures 152

of Morris and Dodds (34). Analysis of the dsRNA extracts and identification of the double- 153

strandedness of RNAs were done with the methods described in our previous studies (51). 154

cDNA cloning, sequencing and sequence analysis. The dsRNAs extracted from strain 155

Bc-72 of B. porri were fractioned by agarose gel electrophoresis and the individual dsRNA bands 156

(6.5 kb for dsRNA-1 and 6.2 kb for dsRNA-2) were cut off for further analysis. Cloning of the 157


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cDNAs for the dsRNAs from strain Bc-72 by random primers-mediated PCR, sequencing of the 158

cDNAs and analysis of the sequences were done using the procedures described in our previous 159

studies (51, 52). To clone the terminal sequences of each dsRNA, the 3’-terminus of each strand 160

of dsRNA-1 or dsRNA-2 was ligated to the 5’-terminus of the BPA adaptor (Fig. S1 and Table 161

S1 in Supplemental material) using T4 RNA ligase (TAKARA Biotechnology Co., Ltd., Dalian, 162

China) at 4–8 ºC for 18 h. The primer RCBPA was used for reverse transcription of dsRNA-1 or 163

dsRNA-2 to cDNA. The primer pairs RCBPA/BP5-4 and RCBPA/BP3 were used for 164

PCR-amplification of the 5’-terminal sequence and the 3’-terminal sequence of dsRNA-1, 165

respectively. Similarly, the primer pairs RCBPA/BG1 and RCBPA/BG2 were used for 166

PCR-amplifying the 5’-terminal sequence and the 3’-terminal sequence of dsRNA-2, respectively 167

The PCR primers and their sequences mentioned above are listed in Table S1 (in Supplemental 168

material). The full-length cDNA sequence for dsRNA-1 or dsRNA-2 was obtained by assembling 169

of the partial sequences in the different cDNA clones (Fig. S1 in Supplemental material). Open 170

reading frames (ORFs) in each full-length cDNA sequence were deduced using the ORF Finder 171

program in the website of the National Center for Biotechnology Information (NCBI) 172

(http://www.ncbi.nlm. nih.gov/gorf/ gorf.html) with the standard codon usages. Database 173

searches of the full-length cDNA sequences of dsRNA-1 and dsRNA-2 and deduced polypeptides 174

from the two cDNA sequences were performed in the public database at NCBI using the 175

programs of BLASTn and BLASTp, respectively. Motifs present in the deduced polypeptide 176

sequences were searched in three databases, including the CDD database 177

(http://www.ncbi.nlm.nih.gov/Structure /cdd/wrpsb.cgi), the Pfam database 178

(http://pfam.sanger.ac.uk/) (15) and the PROSITE database (http://www.expasy.ch/). The 179

deduced polypeptide sequences encoded by the two dsRNAs were also BLAST-searched in the 180


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MEROPS protease database (http://merops.sanger.ac.uk/) (40). Multiple alignments of the 181

sequences of RNA-dependent RNA polymerase (RdRp) was accomplished using the 182

CLUSTAL_X program (48). A phylogenetic tree was constructed using the neighbor joining (NJ) 183

method and tested with a bootstrap of 1,000 replicates to ascertain the reliability of a given 184

branch pattern in the NJ tree. Potential secondary structures at the 3’- and 5’-terminal sequences 185

of the positive strand of dsRNA-1 and dsRNA-2 were predicted using the RNAstructure software 186

(version 4.6) (32). The proline frequency on the two polypeptides deduced from dsRNA-ORF I 187

on dsRNA-1 and ORF II on dsRNA-2 was calculated using Microsoft Office Excel 2007 as 188

described by Spear et al. (46). The threshold value of 0.33 was used to indicate the minimum 189

proline frequency required for generation a PPII-like helix (PxxPxxPxx) over a nine-amino acids 190

span. 191

Northern hybridization. Northern hybridization was performed to verify the authenticity of 192

the cDNA sequences generated from dsRNA-1 (6.5 kb) and dsRNA-2 (6.2 kb) in strain Bc-72 of 193

B. porri. Meanwhile, it was also used to confirm the high nucleotide sequence similarity between 194

the two dsRNAs at the 5’-terminus (Fig. 1A). The dsRNAs from strain Bc-72 were separated by 195

agarose gel electrophoresis. They were then transferred from the agarose gel to an 196

Immobilon™-Ny+ membrane (Millipore, Bedford, MA, USA) using the procedures described by 197

Jiang and Ghabrial (25). Three DNA probes (423 bp for probes 1, 506 bp for probe 2 and 451 bp 198

for probe 3) were designed on the basis of the full-length cDNA sequences of dsRNA-1 and 199

dsRNA-2. They were generated by reverse-transcription PCR (RT-PCR) using dsRNA-1 or 200

dsRNA-2 as template with the specific primer pairs listed in Table S1 (in Supplemental material). 201

The probes were individually labelled with digoxigenin (DIG)-11-dUTP in the DIG High Prime 202

DNA Labeling and Detection Starter Kit I (Roche Diagnostics GmbH, Roche Applied Science, 203


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Mannheim, Germany) and used to hybridize with the dsRNAs blotted on the nylon membrane. 204

The hybridization signals were detected by enzymatic immunoassay using the reagents and the 205

procedures recommended by the manufacturer (Roche Applied Science). 206

Purification of viral particles. Strain Bc-72 of B. porri was grown at 20 ºC for 6 days on 207

sterilized cellophane films placed on PDA. Mycelia were harvested and a sample of 10 g fresh 208

mycelia was ground to fine powder in liquid nitrogen using sterilized mortar and pestle. The 209

powder was transferred to a 1,000 mL-blender containing 150 mL extraction solution [0.1 M 210

sodium phosphate and 3% (w/v) Triton X100, pH 7.0]. The suspension was blended for 30 s and 211

then transferred to a plastic tube (200 mL), followed by centrifuging at 10,000 × g for 20 min to 212

remove the hyphal cell debris. The supernatant was then carefully pipetted out to another plastic 213

tube, which was then subject to a 2-h-long ultra-centrifuge at 119,000 × g under 4 ºC to 214

precipitate all the particles including the virus particles in the supernatant. The pellet was 215

re-suspended in 4 mL sodium phosphate buffer (0.1 M) and the resulting suspension was 216

centrifuged at 16,000 × g for 20 min to precipitate large particles. Then, the supernatant 217

containing the virus particles was overlayed on a centrifuge tube containing sucrose solutions 218

with a concentration gradient ranging from 10 to 40% (w/v) and centrifuged at 70,000 × g under 219

4 ºC for 2 h. Fractions in the middle portion of the tube were individually measured for the 220

presence of the virus particles by detection of the dsRNA segments using the agarose gel 221

electrophoresis. The fraction containing virus particles was carefully collected and suspended in 222

100 µL sodium phosphate buffer (0.05 M, pH 7.0). The virus particles were stained with 223

phosphotungstic acid solution [20 g/L (w/v), pH 7.4] and observed under TEM. dsRNA from 224

viral particles was sequentially extracted with phenol, phenol-chloroform, and 225

chloroform-isoamyl alcohol and fractionated by electrophoresis on an agarose gel (1%, w/v). The 226


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SPs from viral particles were detected by electrophoresis on a polyacrylamide gel (12%, w/v) 227

amended with sodium dodecyl sulfate (SDS) (1%, w/v). 228

Peptide mass fingerprinting (PMF). The virus particle suspension, derived from B. porri 229

strain Bc-72 was loaded on a 12% polyacrylamide gel amended with 1% SDS. After 230

electrophoresis, the gel was stained with Coomassie brilliant blue G250. The protein bands on the 231

gel were individually excised to make different gel samples. Each gel sample was sequentially 232

destained with NH4HCO3 (100 mM) and acetonitrile (ACN) (30%, w/v). The gel samples were 233

lyophilized, followed by rehydration in 5 µL NH4HCO3 solution (50 mM) amended with 50 ng 234

trypsin (Sequencing Grade, Promega, Madison, WI, USA). The mixtures in small tubes were 235

bath-incubated at 37 ºC overnight to digest the proteins. The resulting peptides from each gel 236

sample were extracted three times with 1 mL of extraction buffer containing 0.1% trifluoroacetic 237

acid (TFA, w/v) and 60% ACN. The aqueous phase of the extracts for each sample was pooled 238

and lyophilized. The lyophilized peptides mixture for each protein was dissolved in 0.1% TFA. 239

The resulting solution was desalted and concentrated using ZipTip® pipette tips (Millipore 240

Bedford, MA, USA). Then, the peptides were dissolved in R-cyano-4-hydroxycinnamic acid (5 241

mg/mL) amended with 0.1% TFA and 50% ACN and analyzed by matrix-assisted laser 242

desorption/ionization–time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF 243

tandem MS on a 4800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems, Foster City, 244

USA). Molecular weight of each peptide was compared to that deduced from the nucleotide 245

sequences of ORF I on dsRNA-1 or ORF II on dsRNA-2 using the MASCOT program (Matrix 246

Science Ltd., London, UK). 247

Protoplast transfection. The virus-free strain Bc-38 of B. porri was used as the recipient in 248

this experiment. Hypovirulence-associated dsRNAs in strain Bc-72 could not be transmitted to 249


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from strain Bc-72 to strain Bc-38 in the horizontal transmission experiment of this study. 250

Protoplasts were prepared from actively-growing mycelia of strain Bc-38 using the procedures 251

described by Yelton et al. (54). The method described by Kanematsu et al. (27) was used to 252

transfect protoplasts of strain Bc-38 with the purified virus particles from strain Bc-72 in the 253

presence of polyethylene glycol (PEG) 4000. The transfected protoplast suspension was 254

incubated without shaking at 20 °C in the dark for 2 days and plated on YCGA media (0.1% 255

yeast extracts, 0.1% casein hydrolysate, 0.5 M glucose and 1.5% agar in Petri dishes (9 cm 256

diameter), this was followed by incubation at 20 °C for 3 days. Agar plugs containing mycelia 257

generated from the protoplasts were individually transferred to PDA plates, one plug per plate. 258

The dishes were incubated at 20 °C in the dark for 12 days, and the resulting cultures were 259

identified and screened for abnormal colonies (slow growth and formation of mycelial sectors). A 260

representative virus-transfected isolate 38T was tested for mycelial growth rate (20 °C) on PDA, 261

pathogenicity on detached garlic leaves (20 °C, 72 h) and the presence of the dsRNAs in mycelia. 262

The parental strain Bc-38 was used as a control strain in these tests. 263

Statistical analysis. Data on mycelial growth rate and leaf lesion length caused by strains of 264

B. porri were analyzed using analysis of variance (ANOVA) in SAS V8.0 (SAS Institute, Cary, 265

NC, USA, 1999). Treatment means on each of these two parameters for the tested strains/isolates 266

were compared using the Least Significant Difference Test at α = 0.05. Significant differences 267

between strains Bc-38 and 38T of B. porri in each of the two parameters were detected using 268

Student’s t-test at α = 0.01. 269

RESULTS 270

Virus particles. The virus particles purified from strain Bc-72 of Botrytis porri were 271

isometric and ~35 nm in diameter (Figs. 2A, B). The virus particles accommodate two dsRNA 272


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segments with similar sizes to dsRNA-1 and dsRNA-2 extracted from mycelia of strain Bc-72 273

(Fig. 2C). SDS/PAGE of the purified particles revealed the presence of three major proteins with 274

estimated molecular mass of 70, 80 and 85 kDa designated as p70, p80 and p85, respectively 275

(Fig. 2D). It is worth noting that the virion dsRNA segments showed similar brightness to the two 276

dsRNAs extracted from the mycelia, suggesting that the two dsRNAs occur at the similar molar 277

ratios in both cases. 278

Nucleotide sequences of dsRNA-1 and dsRNA-2. A total of 21 cDNA clones were 279

obtained using random hexameric primers. Assembly sequences of these cDNA clones generated 280

two contigs. Contig 1 comprised 13 clones belonging to dsRNA-1 and contig-2 comprised 8 281

clones belonging to dsRNA-2 (Fig. S1 in Supplemental material). No other cDNA clones were 282

found to make additional contigs, suggesting that no other dsRNAs in strain Bc-72 have the 283

similar size to dsRNA-1 or dsRNA-2. The full-length cDNA sequences for dsRNA-1 (GenBank 284

Acc. No. JF716350) and dsRNA-2 (GenBank Acc. No. JF716351) were 6,215 and 5,879 bp in 285

length, respectively (Fig. 3). Interestingly, we observed that the nucleotide sequences of 286

dsRNA-1 and dsRNA-2 at the 3’-termin (80-bp long) and the 5’-termini (500-bp long) shared 287

sequence identities of 62 and 95%, respectively (Fig. 1A, B). Moreover, the extreme terminal 288

sequences of dsRNAs are strictly conserved. Results of the Northern hybridization analysis 289

confirmed the high nucleotide sequence identity at the 5’-termini of dsRNA-1 and dsRNA-2 (Fig. 290

3A). Probe 1 (423 bp) corresponding to sequences at the 5’-terminus of dsRNA-1 simultaneously 291

hybridized with both dsRNA-1 and dsRNA-2. However, probe 2 (506 bp) corresponding to 292

sequences at the middle region of dsRNA-1 hybridized only with dsRNA-1, but did not hybridize 293

with dsRNA-2. Similarly, probe 3 (451 bp) corresponding to sequences at the middle region of 294

dsRNA-2 hybridized only with dsRNA-2. 295


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The 5’- and 3’-untranslated regions (UTR) of dsRNA-1 and dsRNA-2 were examined for 296

potential secondary structures using the RNA structure software (version 4.6) (32). Results 297

showed that formation of a stem-loop structure was detected at the 3’- and 5’-terminal regions of 298

dsRNA-1, and at the 5’-terminal region of dsRNA-2, but was not detected at the 3’-terminial 299

region of dsRNA-2 (Fig. 1C). Results also showed that both the 3’- and 5’-terminal regions of 300

dsRNA-1 and dsRNA-2 lack inverted complementarity required for formation of any panhandle 301

structures. 302

Analysis of the putative polypeptides. Both strands of the full-length cDNAs of dsRNA-1 303

and dsRNA-2 were examined for open reading frames (ORFs). Two ORFs, designated as ORF I 304

and ORF II, were found on the positive strand of dsRNA-1 (404–6112) and dsRNA-2 305

(405–5771), respectively (Fig. 3B). ORF I was deduced to encode a polypeptide with 1902 amino 306

acids. It has a proline-rich region at positions 221–264 (Fig. S2 in Supplemental material), 307

corresponding to the nucleotide (nt) position at 1067–1196 (Fig. 3B). It also contains 308

RNA-dependent RNA polymerase (RdRp)_4 surperfamily domain at nt positions 4625–5455 309

(Fig. 3). The predicted RdRp sequence includes the eight conserved motifs (I to VIII) similar to 310

the RdRp sequences of viruses in the families Totiviridae, Chrysoviridae, Megabirnaviridae and 311

a few unassigned virus taxa (Fig. 4). Therefore, dsRNA-1 and dsRNA-2 are proposed as the 312

genome segments of a mycovirus. We designated this mycovirus as Botrytis porri RNA virus 1 313

(BpRV1). Results from BLASTp analysis showed that RdRp sequence has low levels of 314

sequence similarity (< 25%) to the RdRp sequences of viruses in the families Totiviridae, 315

Chrysoviridae, Megabirnaviridae and as well as to those of a few unassigned virus taxa (Table 316

2). With the exception of the RdRp sequence, no proteins homologous to the remaining part of 317

ORF I-encoded polypeptide were identified in the database of NCBI. Moreover, the polypeptide 318


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of ORF I was searched for homologous proteases in the MEROPS protease database. However, 319

no hits with E values less than 0.01 were detected. 320

ORF II on dsRNA-2 was deduced to encode a polypeptide with 1788 amino acids (Fig. 3B). 321

The polypeptide sequence was detected to contain numerous phosphorylation/glycosylation sites. 322

No conserved proteins homologous to this polypeptide were found in the database of NCBI. 323

Moreover, no hits indicating the presence of any known homologous proteases on this 324

polypeptide were detected in the MEROPS protease database. 325

The three proteins (p70, p80 and p85) detected in the purified virus particles of BpRV1 were 326

individually cleaved with trypsin and the resulting peptide fragments for each protein were 327

subject to PMF analysis. Results showed that p70, p80 and p85 generated a total of 13, 23 and 22 328

peptide fragments, respectively (Tables S2, S3, S4 in Supplemental material). Of these peptide 329

fragments, 11 from p70 matched the partial peptide sequence encoded by ORF II at amino acid 330

positions 200–850, accounting for 30.7% of the entire coverage (651 amino acids). Four peptide 331

fragments matched the deduced sequences HSVNLWSYRPTSAQSR (319–334), 332

AVNTGIGYTSPR (337–348), TDLSIVPTYYTYQTDIR (505–521) and YLEETFTTGR 333

(714–723), with the ion scores higher than 46 (> 95% confidence) (Table S4 in Supplemental 334

material). Twenty one peptide fragments from p80 and 18 peptide fragments from p85 matched 335

the partial peptide sequence encoded by ORF I at amino acid positions 200–1050 (amino acid 336

position) accounting for 41.9 and 30.7% of the entire coverage (851 amino acid residues), 337

respectively. Seven peptide fragments from these two proteins matched the deduced sequences, 338

including DTIAYNVPFGMSVYNR (407–422), FLLPVEDGDDAPIRK (501–515), 339

ITTYVTAGPGAPDLAVHSTIDLYVPEQR (552–579), FTLCPLNGSELIEER (580–594), 340

YLTAHNLWQQFPVMR (616–630), QILQQEYCTER (807–817), and 341


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VAQENVGAVTYYQHEILPSESVSDTPVVR (829–857), with the ion scores higher than 46. 342

Therefore, p80 might be derived from p85 via post-translational modification in a comparable 343

manner to the related capsid proteins of the victorivirus Helminthosporium victoriae virus 190S 344

(HvV190S) (24). 345

Phylogenetic analysis. A phylogenetic tree based on RdRp sequences of BpRV1 and 38 346

selected RNA viruses was generated using the neighbor joining method (Fig. 5). As no members 347

of the reported bipartite dsRNA viral families Birnaviridae, Partitiviridae and Picobirnaviridae 348

were indentified through BLAST search (Table 2), these viruses were not selected to establish the 349

phylogenetic tree. Phylogenetic analysis revealed that BpRV1 appears to be more closely related 350

to members of the genera Totivirius and Victorivirus (both in the family Totiviridae) than to 351

members of the families Chrysoviridae and Megabirnaviridae. BpRV1 clustered with two 352

unassigned insect viruses, Spissistilus festinus virus 1 (SFV1) and Cirulifer tenellus virus 1 353

(CiTV1), forming the BpRV1-SFV1-CiTV1 clade, which was more closely related to Ustilago 354

maydis virus H1 (UmVH1) than to ScV-L-A and ScV-LBC, forming the 355

BpRV1-SFV1-CiTV1-UmVH1 clade. However, the bootstrap support for generation of the two 356

clades was weak, 408 and 343 (1,000 replicates) for support of the clades of 357

BpRV1-SFV1-CiTV1 and BpRV1-SFV1-CiTV1-UMVH1, respectively. 358

Hypovirulence, abnormal colony morphology and dsRNAs. Strains Bc-16, Bc-38 and 359

Bc-95 of B. porri, which lack detectable dsRNAs, caused large necrotic lesions on garlic leaves 360

(20 ºC, 72 h) with the average lesion length of 12 to 17 mm (Fig. 6A). On PDA, they grew at 361

radial growth rates ranging from 11.4 to 13.2 mm/d under 20 ºC (Fig. 6B), and formed 362

grey-whitish colonies and black cerebriform sclerotia after incubation for 17 days (Fig. 6C). In 363

contrast, strain Bc-72 of B. porri harboring two dsRNA segments caused small lesions on garlic 364


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leaves with the average lesion length of 2.3 mm (Fig. 6A). On PDA, it grew at a radial growth 365

rate of 2.3 mm/d under 20 ºC (Fig. 6B) and produced grey-brownish colonies with the formation 366

of numerous mycelial sectors at the colony margin (Fig. 6C). No sclerotia were formed on the 367

colonies of strain Bc-72 after incubation for 17 days (Fig. 6C). 368

Hyphae of strains Bc-16 and Bc-72 were sampled both from the colony margin area and 369

from the colony centre area. Ultra-thin hyphal sections for each strain were observed under TEM. 370

The cytoplasm of hyphal compartments in strain Bc-72 showed a degeneration syndrome 371

manifested by formation of abundant vacuole-like membranous structures and small membranous 372

vesicles (Fig. 7A, B, C). In most cases, the membranous vacuoles/vesicles aggregated and 373

merged. In contrast, the cytoplasm of hyphal compartments of comparable age from strain Bc-16 374

was densely stained and evenly-distributed without formation of abundant and aggregated 375

membranous vacuoles/vesicles (Fig. 7D). Moreover, virus-like particles with the similar size to 376

the virus particles of BpRV1 were observed in the hyphal cells of Bc-72 (Fig. 8). 377

Vertical transmission of hypovirulence-associated dsRNAs. Thirty-five single-conidial 378

(SC) isolates derived from strain Bc-72 were obtained. Thirty-four SC isolates (GP72SC01 to 379

GP72SC34) were detected to be BpRV1-positive and only one SC isolate named GP72SC35 380

(shortened as SC35) was detected to be BpRV1-negative (Fig. 9). On PDA, all the 381

BpRV1-postive SC isolates grew at the average mycelial growth rates ranging from 1.0 to 6.4 382

mm/d, significantly lower (P < 0.05) than the BpRV1-free strain Bc-16 and isolate SC35 (9.9 383

mm/d). On garlic leaves, they produced no lesions or small lesions with the average length 384

ranging from 0 to 3.6 mm, significantly shorter (P < 0.05) than the average lesion length for 385

strain Bc-16 (15.1 mm) and isolate SC35 (11.7 mm). We also found that the average lesion 386

length caused by the BpRV1-positve SC isolates were not significantly correlated with the 387


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average mycelial growth rates of these isolates (r 2 = 1 × 10-7, P > 0.01). Therefore, BpRV1 in 388

strain Bc72 can be vertically transmitted from mycelia to conidia and the hypovirulence 389

phenotype appears just qualitatively correlated with the transmission of BpRV1. 390

Horizontal transmission of hypovirulence-associated dsRNAs. Results of the horizontal 391

transmission experiment showed that in the single cultures, strains Bc-38, Bc-95 and SC35 grew 392

rapidly on PDA and colonized the entire dishes after incubation for 8 days (20 ºC), whereas strain 393

Bc-72 grew slowly and formed small abnormal colonies (Fig. 10). In the contact cultures of 394

Bc-72/Bc-38 and Bc-72/Bc-95, strains Bc-38 and Bc-95 still grew rapidly and colonized the 395

entire dishes after 8 days. In the contact cultures of Bc-72/SC35, strain SC35 grew rapidly in the 396

first three days, followed by reduced growth and formation of mycelial sectors at the colony 397

margin (Fig. 10). However, strain SC35 still failed to colonize the entire dishes in the contact 398

cultures of Bc-72/SC-35 after incubation for 8 days. 399

Three mycelial derivative isolates of strain SC35 (72-35-1, 72-35-2 and 72-35-3) were 400

obtained from three recipient colonies of SC35 in the three contact cultures of Bc-72/SC35 (Fig. 401

10). They were similar to strain Bc-72 in mycelial growth on PDA with the average growth rates 402

of 2.2–3.4 mm/d and in pathogenicity on garlic leaves with the average leaf lesion length of 403

1.1–2.4 mm (Fig. S3 in Supplemental material). However, these derivatives differed greatly from 404

their parental strain SC35, which grew at 10.2 mm/d on PDA and caused lesions of 12.3 mm in 405

length on garlic leaves. Both dsRNA-1 and dsRNA-2 were detected in 72-35-1, 72-35-2 and 406

72-35-3, as well as in strain Bc-72, but were not detected in strain SC35. Therefore, BpRV1 in 407

strain Bc-72 can be horizontally transmitted to strain SC35 through hyphal contact and the 408

hypovirulence phenotype appears qualitatively correlated with virus transmission. 409


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Three mycelial derivative isolates of strain Bc-38 (72-38-1, 72-38-2 and 72-38-3) were 410

obtained from the three recipient colonies of strain Bc-38 in the contact cultures of Bc-72/Bc-38 411

(data not shown). Three mycelial derivatives of strain Bc-95 (72-95-1, 72-95-2 and 72-95-3) were 412

obtained from the three recipient colonies of strain Bc-95 in the contact cultures of Bc-72/Bc-95 413

(Fig. 10). All these mycelial derivative isolates were similar to the corresponding parental strains 414

Bc-38 and Bc-95 both in mycelial growth rate on PDA (20 ºC) and in leaf lesion length on garlic 415

leaves (Fig. S3 in Supplemental material). No dsRNA was detected in these derivative isolates, as 416

well as in their parental strains Bc-38 and Bc-95. Therefore, BpRV1 in strain Bc-72 can not be 417

horizontally transmitted to strains Bc-38 or Bc-95 through hyphal contact. 418

Virulence attenuation as a consequence of transfection with purified virions. The 419

contact culture experiments aiming at co-transmission of the hypovirulence trait and the 420

hypovirulence-associated dsRNAs from strain Bc-72 to strain Bc-38 (BpRV1 free) was 421

unsuccessful (Fig. S3 in Supplemental material). As an alternative, we tried to transfect 422

protoplasts of strain Bc-38 with the purified BpRV1 particles in the presence of PEG 4000. A 423

derivative isolate 38T was obtained from the generated protoplasts of strain Bc-38. It grew at the 424

average radial rate of 4.3 mm/d on PDA at 20 °C with formation of abnormal whitish compacted 425

colonies (Fig. 11A) and was hypovirulent on garlic leaves with the average lesion length of 3.3 426

mm (20 °C, 72 h) (Fig. 11). Two dsRNA segments present in strain Bc-72 were consistently 427

detected in the mycelia of 38T (data not shown). The identity of the dsRNAs in 38T as the 428

genome of BpRV1 was confirmed using RT-PCR with the BpRV1-specific primers 429

BVP1F/BVP1R (Table S1 in Supplemental material). 430

DISCUSSION 431


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The present study reveals that hypovirulence of strain Bc-72 of Botrytis porri is caused by 432

infection with a novel bipartite dsRNA mycovirus (BpRV1). To our knowledge, this is the first 433

report about the co-occurrence of a dsRNA mycovirus and hypovirulence in B. porri. This study 434

provided three lines of experimental evidence supporting the conclusion that BpRV1 is the causal 435

agent of hypovirulence in B. porri. The first evidence is based on the vertical transmission 436

experiment. Thirty-four BpRV1-infected single-conidium (SC) isolates of strain Bc-72 were 437

hypovirulent on garlic leaves, whereas the BpRV1-free SC isolate SC35 became virulent on 438

garlic leaves (Fig. 9). The second evidence is derived from the horizontal transmission 439

experiment, in which BpRV1 was transmitted to the virus-cured strain SC35 through hyphal 440

contact (Fig. 10). The three derivative isolates of strain SC35 showed reduced mycelial growth 441

rates on PDA and debilitated virulence on garlic leaves (Fig. S3 in Supplemental material). The 442

third evidence is from the transfection experiment, in which the purified BpRV1 particles were 443

successfully introduced into the protoplasts of the BpRV1-free virulent strain Bc-38 of B. porri. 444

The transfected derivative isolate 38T showed debilitation symptoms, including reduced mycelial 445

growth on PDA and hypovirulence on garlic leaves (Fig. 11A). 446

It is of interest that the 34 BpRV1-infected hypovirulent SC isolates of strain Bc-72 varied 447

greatly both in mycelial growth rates (1.0 to 6.4 mm/d) on PDA and in leaf lesion length (0 to 3.6 448

mm) on garlic leaves. We interpret this kind of variation as inter-isolate variation, which might be 449

caused by different levels of virus accumulation in different SC isolates. We also observed that 450

there was no significant correlation (P > 0.01) between the two parameters. This result might be 451

due to the unstable cultural characteristics for some SC isolates. We call this kind of variation as 452

intra-isolate variation, which might be caused by the uneven distribution of BpRV1 in hyphal 453

cytoplasm. We observed that the subcultures of even the same SC isolate differed greatly in 454


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colony morphology on PDA, colonies of some subcultures showing normal appearance (slightly 455

attenuated) and colonies of some subcultures showing very abnormal appearance (severely 456

attenuated) (M.D. Wu and G.Q. Li, unpublished data). The intra-isolate variability may mask or 457

obscure the correlative relationship between the mycelial growth rates and the leaf lesion length 458

values. We also observed that the hypovirulent isolate 38T infected by BpRV1 (protoplast 459

infection) showed dramatic differences from the hypovirulent strain GarlicBc-72 and its 460

hypovirulent derivatives, such as 72-35-1 (Fig. 11) in colony morphology on PDA, although 461

mycelial growth of all these hypovirulent isolates was impaired. The difference between the 462

hypovirulent strains 38T and 72-35-1 in colony morphology might be caused by different genetic 463

backgrounds. 464

Results of the horizontal transmission experiment showed that co-transmission of the 465

hypovirulence trait and BpRV1 was successful from strain Bc-72 to virulent strain SC35 of B. 466

porri, but was unsuccessful from Bc-72 to two other virulent strains (Bc-38 and Bc-95) of B. 467

porri. Therefore, the strain-specific transmission manner appears similar to the horizontal 468

transmission of Botrytis cinerea mitovirus 1 (BcMV1) (51, 52). The unsuccessful horizontal 469

transmission of BpRV1 might be due to the vegetative incompatibility between the hypovirulent 470

strain Bc-72 and virulent strains Bc-38 and Bc-95. Strain SC35, a single-conidium isolate of 471

strain Bc-72, is isogenic to strain Bc-72 and they may have the same or similar alleles at the 472

vegetative incompatibility (vic) loci, conferring a compatible interaction between hyphal cells of 473

strains Bc-72 and SC35. In contrast, strains Bc-38, Bc-72 and Bc-95 were collected from 474

different geographic locations in China (Table 1). They might have different alleles at the vic 475

loci, thus maybe conferring an incompatible interaction between hyphal cells and failure of 476

BpRV1 transmission. 477


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Previous studies showed that it is often difficult to establish the etiology of a mycovirus as 478

an agent for hypovirulence in phytopathogenic fungi due to the lack of a proper infection method 479

to complete the Koch’s postulates (9). Two approaches have been successfully used to solve this 480

problem. The first approach is to prepare virus-derived cDNA transcripts (7) or to construct 481

infectious viral cDNA clones (10, 28). Both the synthetic cDNA transcripts and the infectious 482

viral cDNA clones can initiate mycovirus infection in host fungi. This approach has been 483

successfully applied in the hypovirus-C. parasitica system to ascertain the determinant role of 484

hypovirus in hypovirulence of C. parasitica (7, 10, 28). Another approach is to transfect cells or 485

protoplasts of host fungi with purified virus particles. A PEG-mediated transfection method with 486

virus particles has been developed for a mycovirus in B. cinerea (6), mycoreviruses in 487

Cryphonectria parasitica (20) and in Rosellinia necatrix (43), a megabirnavirus (9) and a 488

partitivirus (42) in R. necatrix, and an ssDNA mycovirus in Sclerotinia sclerotiorum (55). In this 489

study, we successfully introduced the purified BpRV1 particles into virus-free strain Bc-38 (Fig. 490

11). This provides an additional example showing the potential of using the PEG-mediated 491

mycovirus transfection technique to study the etiology of mycoviruses for fungal hypovirulence. 492

This technique might even be useful for determining the experimental host range of BpRV1 in 493

the future. 494

Sequence analysis indicates that dsRNA-1 and dsRNA-2 in strain Bc-72 comprise the 495

genome of a bipartite virus. Only dsRNA-1 encodes an RdRp, implying that the two dsRNAs are 496

not derived from two independent mycoviruses. Previous studies showed that many multipartite 497

RNA viruses have conserved terminal regions (9, 25, 49). This phenomenon was observed in the 498

two dsRNAs of BpRV1, which has conserved terminal regions (Fig. 1). This result suggests that 499

dsRNA-2 of BpRV1 may depend on the RdRp encoded by ORF I on dsRNA-1 of BpRV1 for 500


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replication. We observed that the remaining regions on the two dsRNAs of BpRV1 have low 501

sequence similarity (< 40%), implying that dsRNA-2 is not a defective RNA of dsRNA-1. In 502

addition, the genome size of BpRV1 appears different from that of other reported bipartite 503

dsRNA viruses, including those in the Birnaviridae (hosts: vertebrates and invertebrates), 504

Partitiviridae (hosts: fungi and plants), Picobirnaviridae (hosts: vertebrates) and 505

Megabirnaviridae (hosts: fungi). The genome size of BpRV1 is ~12 kb, smaller than that of 506

Megabirnaviridae (~17 kb) (9), but larger than those of Partitiviridae (2.8–4.6 kb) (16), 507

Picobirnaviridae (~4 kb) (9) and Birnaviridae (~6 kb) (9). 508

The RdRp of BpRV1 contains eight conserved motifs, which appear similar to the conserved 509

motifs of the viral RdRps in Totiviridae, Chrysoviridae, Megabirnaviridae and a few 510

taxonomically-unassigned viruses, including SFV1 and CiTV1 (Fig. 4), but are different from the 511

RdRp motifs of other bipartite viruses (Birnaviridae, Partitiviridae and Picobirnaviridae). 512

However, the RdRp sequence of BpRV1 shows low sequence similarity levels (< 25%) to the 513

RdRp sequences of the mycoviruses in Totiviridae, Chrysoviridae and Megabirnaviridae (Table 514

2). In the phylogenetic tree inferred from the viral RdRp sequences (Fig. 5), BpRV1 formed a 515

separate clade distinct from those for Totiviridae, Chrysoviridae, Megabirnaviridae and a few 516

taxonomically-unassigned virus taxa. It has a closer phylogenetic relationship to SFV1, CiTV1 517

and UmVH1 than with RnMBV1. However, BpRV1 with two dsRNA segments differs from 518

these three viruses in the number of dsRNA segments since genomes of SFV1, CiTV1 and 519

UmVH1 are monosegmented (26, 46). Although BpRV1 and RnMBV1 have bipartite dsRNA 520

genomes and their RdRps share sequence similarities, BpRV1 differs from RnMBV1 in the 521

expression strategy for generation of RdRp and the structural proteins. For BpRV1, the RdRp of 522

BpRV1 is more likely expressed as a fusion protein containing a part of the structural proteins 523


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(p80/p85) (Fig. 3). The other structural protein component (p70) is more likely encoded by ORF 524

II. However, RnMBV1 has two ORFs on dsRNA-1, ORF 1 for capsid protein and ORF 2 for 525

RdRp (9). 526

Taking together the information about the number of dsRNA segments, the RNA genome 527

size, the genetic organization and the RdRp sequences, we believe that BpRV1 is a novel 528

bipartite dsRNA mycovirus, which can not be assigned to any known mycovirus families. 529

Furthermore, BpRV1 has been detected in a strain of Botrytis squamosa Walker, the causal agent 530

of onion leaf blight (M. D. Wu, unpublished data). The BpRV1-infected strain of B. squamosa 531

was collected from Tongshan County of Hubei Province, China, ~500 kilometers away from 532

Zhushan County, where strain Bc-72 of B. porri was originally collected. Moreover, a bipartite 533

dsRNA mycovirus infecting Sclerotinia sclerotiorum, the causal agent of sclerotinia stem rot of 534

oilseed rape, was recently identified in China. The sequences of the two dsRNAs were identical 535

to dsRNA-1 and dsRNA-2 of BpRV1 by 91 and 97%, respectively (L. J. Liu and D. H. Jiang, 536

unpublished data). These results suggest that besides B. porri, BpRV1 can infect B. squamosa 537

and S. sclerotiorum. It might be widely distributed in B. porri-, B. squamosa- and S. 538

sclerotiorum-occurring regions. 539

BpRV1 particles are approximately 35 nm in diameter. The size is similar to that of the 540

families Chrysoviridae (30 to 40 nm), Partitiviridae (30 to 40 nm) and Totiviridae (30 to 40 nm), 541

but is smaller than that of RnMBV1 in Megabirnaviridae (~50 nm) and Mycoreovirus (~80 nm). 542

Whether or not the two dsRNAs of BpRV1 are encapsidated in separate virus particles or within 543

the same virus particles remains unknown. The dsRNA segments for the genomes of the viruses 544

in Chrysoviridae and Partitiviridae are separately encapsidated (17, 18, 41). In addition, the two 545

dsRNA segments for RnMBV1 were thought to be separately encapsidated based on the finding 546


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that the unequal molar ratios between dsRNA-1 and dsRNA-2 extracted directly from host 547

mycelia and the purified virus particles (9). However, no significant molar ratio variation was 548

observed for BpRV1 dsRNA-1 and dsRNA-2 (Fig. 2C). To address the question about the 549

particle assembly in BpRV1, we calculated the gross volumes of virus particles of dsRNA viruses 550

in all recognized genera based on diameter of virus particles (Table S5 in Supplemental material). 551

Then, the values of the dsRNA length encapsidated within one volume unit of a virus particle 552

were inferred to indicate the dsRNA assembly density. For almost all dsRNA viruses, the density 553

of dsRNA in one particle is within the range of 0.03–0.28 bp/nm3. For BpRV1, the values for the 554

dsRNA density are 0.28 bp/nm3 under separate encapsidation and 0.53 bp/nm3 under combined 555

encapsidation. From this point of view, the two dsRNAs of BpRV1 are likely to be separately 556

encapsidated, as the virus particles might not be spacious enough for simultaneous packaging of 557

the two dsRNAs. 558

Results of the PMF analysis of the three proteins (p70, p80 and p85) in BpRV1 indicate that 559

there may be two structural proteins (SPs) for BpRV1 (Fig. S4 and Tables S2, S3, S4 in 560

Supplemental material). The protein component p70 is encoded by ORF II on dsRNA-2, whereas 561

p80 and p85 are encoded by ORF I at a comparable region on dsRNA-1. There are two 562

possibilities for generation of p80 and p85. The first possibility is that p80 might be derived from 563

p85 via post-translational modification in a comparable manner to the related capsid proteins of 564

HvV190S (24). Another possibility is that p80 and p85 are generated via different events in 565

processing the ORF I-encoded polyprotein. In the PMF analysis, we also found several peptide 566

sequences matched the C-terminal regions, rather than the N-terminal regions, on the ORF I- and 567

ORF II-encoded polypepetides (Fig. S4 in Supplemental material). They are probably random 568


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matches, as the ion scores for these peptide sequences are lower than 46 (Table S2, S3, S4 in 569

Supplemental materials). 570

The genomes of RNA viruses are susceptible to chemical or physical damages from 571

environmental factors. They also encounter immune response or antiviral defense of their hosts. 572

To overcome these challenges, many RNA viruses have developed series of self-defense 573

mechanisms (4). In the interaction between hypoviruses and Cryphonectria parasitica, the 574

hypovirus-encoded papain-like protease p29 acts as an inhibitor of the RNA silencing-induced 575

antiviral defense by C. parasitica, thus enhancing viral RNA accumulation and vertical 576

transmission (44, 45, 47). Moreover, hypovirus infection of C. parasitica causes proliferation of 577

vesicle formation within hyphal cells (11, 35, 36) and these vesicles were determined to be sites 578

for viral replication (13, 14). The replication of RNA viruses in these membranous lipid vesicles 579

might also offer protection from nucleases (31). 580

In addition, Spear et al. (46) proposed that the proline-alanine rich protein (PArp) encoded 581

by SFV1 and CiTV1 may act like a scaffold protein to interact with some membrane components 582

in insect cells, as many animal viruses can utilize a protein scaffold containing proline-rich “late” 583

domain motifs to hijack host proteins involved in the vacuolar protein sorting pathway, resulting 584

in formation of multivesicular bodies (8). In the present study, formation of abundant 585

vacuole-like membranous structures and small membranous vesicles in hyphal cells infected with 586

BpRV1 were observed (Fig. 7) and a proline-rich region (PrR) was detected on putative 587

polypeptide encoded by ORF I on dsRNA-1. Therefore, further studies for elucidation of the 588

function of PrR-containing polypeptide in virus accumulation of BpRV1 and for understanding of 589

the expression strategy of dsRNA-1 and dsRNA-2 are warranted. 590

ACKNOWLEDGMENTS 591


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This research was funded by the Natural Science Foundation of China (Grant No. 592

31070122). The authors greatly appreciate the kind help of Dr. S. A. Ghabrial of the University 593

of Kentucky (USA) for critical reading of the manuscript and providing useful literature on 594

mycovirus taxonomy. We also thank Dr. N. Suzuki at Okayama University (Japan) in providing a 595

protocol for the transfection experiment with the purified virus particles of BpRV1, and Professor 596

N. Hong of Huazhong Agricultural University (China) for her advice to purify virus particles of 597

BpRV1. 598

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57. Zhang, J., G. Q. Li, and D. H. Jiang. 2009. First report of garlic leaf blight caused by 756

Botrytis porri in China. Plant Disease 93:1216. 757

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2010. Botrytis sinoallii: a new species of the grey mould pathogen on Allium crops in China. 759

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Virology Journal 6:96. 763

764

765

766

767

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Figure legends 769

FIG. 1. Alignment of the 5’-terminal (A) and 3’- terminal (B) sequences of the coding strand of 770

dsRNA-1 and dsRNA-2 of Botrytis porri RNA virus 1 (BpRV1). Black shades are used to 771

highlight the identical nucleotides. The “•” symbol represents the missing nucleotide. The start 772

codon (AUG) at the 5’-terminus for both dsRNA-1 and dsRNA-2 is indicated with a black bar 773

above the two nucleotide sequences; (C) The predicted secondary structures for the terminal 774

regions of dsRNA-1 and dsRNA-2 of BpRV1. 775

FIG. 2. BpRV1 particles from strain GarlicBc-72 of Botrytis porri. (A) and (B) Transmission 776

electron microscopic graphs (negative straining) of the virus particles of BpRV1; (C) Agarose gel 777

electrophoresis of the dsRNAs extracted from the purified virus particles of BpRV1, and from 778

mycelia of strains GarlicBc-72 and GarlicBc-38. Marker = 1 kb DNA ladder. Note the similar 779

size for the dsRNAs from the purified virus particles and from the mycelia; (D) SDS-PAGE 780

analysis of the purified virus particles of BpRV1 showing three protein components (p85, p80 781

and p70). 782

FIG. 3. Molecular characteristics of dsRNA-1 and dsRNA-2 of Botrytis porri RNA virus 1 783

(BpRV1). (A) Northern blot detection of dsRNA-1 and dsRNA-2 using digoxigenin-labeled 784

probes 1, 2 and 3. The locations of these probes are indicated in graph B; (B) Schematic diagrams 785

of the genetic organization of BpRV1 dsRNA-1 and dsRNA-2. The coding strand of dsRNA-1 is 786

6,215 bp long and comprises one large open reading frame (ORF), designated as ORF I, which 787

encodes a polyprotein of 1902 amino acid residues. The coding strand of dsRNA-2 is 5,788 bp 788

long and also comprises one large ORF, designated as ORF II, which encodes a polypeptide of 789

1788 amino acid residues. p70, p80 and p85 are the structural proteins of BpRV1 inferred from 790

peptide fingerprinting analyses. 791


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FIG. 4. Multiple alignment of the RdRp sequences of BpRV1 and other RNA viruses showing 792

the structure of the eight-motifs (I to VIII). The alignment was made using CLUSTAL_ X. “*” 793

indicates identical amino acid residues; “:” and “.” indicate high and low chemically-similar 794

amino acid residues, respectively. UmVH1 = Ustilago maydis virus H1 (GenBank Acc. No. 795

NC_003823), ScV-L-A = Saccharomyces cerevisiae virus L-A (J04692), ScV-L-BC = 796

Saccharomyces cerevisiae virus L-BC (La) (U01060), HvV145S = Helminthosporium victoriae 797

virus 145S (AF297176), PcV = Penicillium chrysogenum virus (AF296339), FoCV1 = Fusarium 798

oxysporum chrysovirus 1 (EF152346), AfV = Aspergillus fumigatus chrysovirus (FN178512), 799

CnV1 = Cryphonectria nitschkei chrysovirus 1 (GQ290650), ACDACV = Amasya cherry 800

disease-associated chrysovirus (NC_009947), VcV = Verticillium chrysogenum virus 801

(HM004067), AbV1 = Agaricus bisporus virus 1 (X94361), CCRSCV = cherry chlorotic rusty 802

spot-associated chrysovirus (AJ781397), Hm17V = Helicobasidium mompa no. 17 dsRNA virus 803

(AB085814), SsRV1 = Sphaeropsis sapinea RNA virus 1 (NC_001963), MoV1 = Magnaporthe 804

oryzae virus 1 (AB176964), GaVL1 = Gremmeniella abetina RNA virus L1 (AF337175), BfTV1 805

= Botryotinia fuckeliana totivirus 1 (AM491608), HvV190S = Helminthosporium victoriae virus 806

190S (U41345), RnBMV1 = Rosellinia necatrix megabirnavirus 1 (AB512282), SFV1 = 807

Spissistilus festinus virus 1 (GU979419), CiTV1 = Cirulifer tenellus virus 1 (GU979420), PGV1 808

= Phlebiopsis gigantea mycovirus dsRNA-1 (AM111096), FgV3 = Fusarium graminearum 809

dsRNA mycovirus-3 (NC_013469), DsRV1 = Diplodia scrobiculata RNA virus 1 (NC_013699), 810

PGV2 = Phlebiopsis gigantea mycovirus dsRNA 2 (AM111097), LEV = Lentinula edodes 811

mycovirus HKB (AB429554), PRV = Porcine rotavirus (Q85036), HTV = Human rotavirus 812

(Q91E95), ARV = Avian rotavirus PO-13 (O55590), CMV = Cocksfoot mottle virus (Q66150), 813

RYMV = Rice yellow mottle virus (Q86519), SCMV = Southern cowpea mosaic virus (P21405), 814


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SBMV = Southern bean mosaic virus (O72157), LTSV = Lucerne transient streak virus 815

(Q83093), BWYV = Beet western yellows virus (P09507), CAYV = Cucurbit aphid-borne 816

yellows virus (Q65969), PLV = Potato leafroll virus (P11623), PEMV-1 = Pea enation mosaic 817

virus-1 (P29154). 818

FIG. 5. Phylogenetic analysis of BpRV1 and 38 selected RNA viruses presented in a 819

neighbor-joining (NJ) tree inferred from the RdRp sequences. Animal viruses (rotaviruses) and 820

plant viruses (poleroviruses, enamoviruses and sobemoviruses) with their RdRps belonging to 821

RdRp _4 superfamily were included as outgroups. Numbers at the nodes are bootstrap values out 822

of 1,000 replicates. Bootstrap values lower than 700 are not shown in the figure. The taxonomic 823

status of the viruses labeled with the question mark (?) has not been determined. See the legend 824

for definitions of the virus name abbreviations in FIGURE 4. 825

FIG. 6. Leaf lesion length on garlic, mycelial growth rate on potato dextrose agar and colony 826

morphology of different strains of Botrytis porri. (A) Average lesion length caused by strains 827

GarlicBc-16, GarlicBc-38, GarlicBc-72 and OnionBc-95 on detached garlic leaves (20 °C, 72 h); 828

(B) Average radial growth rates of these strains of B. porri on potato dextrose agar (PDA) at 20 829

°C; and (C) Cultures of strains GarlicBc-16 and GarlicBc-72 on PDA (20 °C, 17 days). In the 830

histograms A and B, results are expressed as arithmetic means ± standard errors of means. Bars 831

in each histogram labeled with the same letters are not significantly different (P > 0.05) 832

according to Least Significant Difference Test. dsRNA– = dsRNA negative, dsRNA+ = dsRNA 833

positive; V = Virulent, HV = Hypovirulent 834

FIG. 7. Transmission electron microscopic graphs showing the cytoplasmic characteristics of the 835

hypovirulent strain GarlicBc-72 and the virulent strain of Botrytis porri. The mycelial samples 836

examined were collected from the colony margin area of each strain. (A, B, C) Three hyphal cells 837


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of strain GarlicBc-72 showing a degeneration sign characterized by formation of abundant 838

vacuole-like membranous structures and small membranous vesicles; (D) A hyphal cell of strain 839

GarlicBc-16 with dense and evenly-distributed cytoplasm; V = vacuoles or vesicles, M = 840

mitochondrion, scale bars = 1 µm. 841

FIG. 8. Transmission electron microscopic graphs of the hyphal cells of the hypovirulent strain 842

GarlicBc-72 containing virus-like particles inside the vesicles (arrowheads). 843

FIG. 9. Lesion length on detached garlic leaves (20 ºC, 72 h) (A) and mycelial growth rates on 844

potato dextrose agar at 20 ºC (B) for strains GarlicBc-16 and GarlicBc-72 of Botrytis porri and 845

35 single-conidium isolates (GP72SC01–GP72SC35) of strain GarlicBc-72. The results were 846

expressed as the arithmetic means ± standard errors of means. In each histogram, bars labeled 847

with the same letters are not significantly different (P > 0.05) according to Least Significant 848

Difference Test. The symbols “+” and “–” indicate the presence and the absence of 849

dsRNA-1/dsRNA-2, respectively. The dsRNAs were extracted from the mycelia of each 850

strain/isolate and detected by agarose gel electrophoresis. 851

FIG. 10. Transmission of the hypovirulence trait and BpRV1 from the hypovirulent strain 852

GarlicBc-72 to the virulent strains GP72SC35 of Botrytis porri on potato dextrose agar. All the 853

dishes were incubated at 20 °C for 8 days. In the single cultures (the top row), strains GP72SC35 854

and OnionBc-95 colonized the entire dishes, whereas strain GarlicBc-72 formed a small colony. 855

In the contact culture of OnionBc-95/GarlicBc-72 (bottom row), OnionBc-95 colonized the entire 856

dish, indicating unsuccessful transmission, as the contact of the hyphae of GarlicBc-72 with the 857

hyphae of GarlicBc-95 had no effect on mycelial growth of GarlicBc-95. However, in the contact 858

culture of GP72SC35/GarlicBc-72, GP72SC35 colonized only a part of the dish with formation 859

of mycelial sector along the colony margin, indicating successful transmission, as the contact of 860


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the hyphae of GarlicBc-72 with the hyphae of GP72SC35 produced a dramatic effect on mycelial 861

growth of GP72SC35. The symbol “*” indicates the place to remove a mycelial agar plug for 862

generation of a derivative isolate for GP72SC35 or OnionBc-95 transfected by BpRV1. 863

FIG. 11. Phenotypic effects of BpRV1 on fungal colony morphology, mycelial growth rate and 864

virulence in strains GarlicBc-38 and GP72SC35 of Botrytis porri. (A) Comparison of the colony 865

morphology between the BpRV1-free strain GarlicBc-38 (left) and the BpRV1-transfected isolate 866

38T (right) (20 °C, 17 days); (B) Comparison of the colony morphology between BpRV1-free 867

isolate GP72SC35 (left) and the BpRV1-infected isolate 72-35-1 (right) (20 °C, 17 days); (C) 868

The mycelial growth rates of strains GP72SC35, 72-35-1, GarlicBc-38 and 38T on PDA (20 °C); 869

(D) Lesion length caused by strains GP72SC35, 72-35-1, GarlicBc-38 and 38Ton garlic leaves 870

(20 °C, 72 h). Results in each histogram are expressed as arithmetic means ± standard errors of 871

means. “**” indicates significant difference (P < 0.01) between strains GarlicBc-38 and 38T, and 872

between strains GP72SC35 and 72-35-1, in mycelial growth rate or lesion length according to 873

Student’s t-test. 874

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GCAAAUAAAAGAGCUGCC .U UUUGAAGCCUUUUAUUUGUAAGGAAAAUUU

AUAUUGAUCGUCAUUCAACGAUCCCCGCUC

GCAAAUAAAAGAGCUGCCUAUUUGAAGCUUUUUAUUUGCAAAGAAAAUGU

AUUUUGAUCGUCAUUAAACGAUCCCCGCUAGGGUUAGAAAGCACCACAAG

CGCAGUGACGATCGCUUGUGAGUAGUAGUCACACUAGAUGGUAAAUUCUA

UUUGGAACCU

GGGUUAGAAAGCGCUACGAA

CGAAGUGACGAACGUACGUAGAUAGUAGUCACACUAAAUGGUAAAUUCUA

UUUGGAACCUCGGACACUCUCAAACCGCCUUGUCAGGAUAUCAGCGGAUG

UGAAUAGACGUGUGGAACACAACGUGCCCACCACAUCUUC

CGGACACUCUCAAACCGCCUUGUCAGGAUAUCAGCGGAUG

UGAAUAGACGUGUGGAACACAACGUGCCCACCACAUCUUCAACUGCUCCU

GUUAGUGUAUAAAUCAGAAGGAUAGGUAAACCGAGAUCUCUAGAGAAG

GCUACCUGGCACUGCUGACG

AACUGCUCCU

GUUAGUGUAUAAAUCAGAAGGAUAGGUAAACCGAGAUCUCUAGAGAAG

GCUACCUGGCACUGCUGACGGGCCGUAGUAUUCUACGAUAUCGGGAGGAA

UUGAAGCCUUCUGUAAUCCCCCAGUACCCAGGCUCUCUAGGGUAAAGUGA

GGCCGUAGUAUUCUACGAUAUCGGGAGGAA

UUGAAGCCUUCUGUAAUCCCCCAGUACCCAGGCUCUCUAGGGUAAAGUGA

GAGAAUGAAAUUCAAAAUGCCUUACAACACACAAACCGCCUCCUCG

AAGAGGACAACAGAGUGGAGAAUGUUGCUA

GAGAAUGAAAUUCAAAAUGCCUUACAACACACAAACCGCCUCCUCG

AAGAGGACAACAGAGUGGAGAAUGUUGCUAAAUCAGCUCCACAAAACAU

AACUUGCCUGCAGAAUUGUG

AAUCAUCUCCACGCAAAA

GCUCAACCUAAGAGCAAGAA

UUACAACAUGCAGUUGGUA..AGUAUAUAGGGUGAUUAGGCUAUCACAUGCAGUGAGGAGUAAAGGAUGAGUUAACCAGG

G.UUAA

CCCCAUAAUUUACAGGGAGAAGUGAAUAGAUAGC

GGCUAA

UCCCAUAUUAUAACGGAAGGAGUAAUUACAGAGC58796215

A

B

dsRNA-1dsRNA-2

AGAG

GCGCGCGC

CCA

The 5’ terminus sequence alignment of the two dsRNA segments

The 3’ terminus sequence alignment of the two dsRNA segments

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

dsRNA-1dsRNA-2

4950

99100

149150

199200

249250

299300

349350

399400

449450

499500

519520

58456181

A UAU

A

GGGU

GAUUA G G

GUU

AACCC

CAUA A

UUUAC

AGGG A

GAA

GUGAAUG

CUUAC

AACAU G

CA

GUUG

GUAAGU AGATAGC

dsRNA-1

10

20

30

GCAAAUAAA

A

GAGCU

GCCUA U

UUG

AAGCUU

UUUAUUUGC

dsRNA-2

5’ terminus 5’ terminus3’ terminusΔG= -13.2 (kcal/mol) ΔG= -16.4 (kcal/mol) ΔG= -14.7 (kcal/mol)

6137

6147

6157

6167

6177

6187 6207

1

10

20

30

GCAAAUAAAAG

AGCU

GCCU U U

UG

AAGC

CUUUUAUUUGU1

C


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200 nm 100 nm

50 nm

GarlicB

c-38

Virus p

articl

es

GarlicB

c-72

Marker

(kb) Virus p

articl

es

Marker

(kDa)

8654

1501201008570

60

p85

A B C D

p80

p70


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404 6112

5’ 3’

62151

405 5771

3’5’1 5879

RdRp_4

4625 5455

dsRNA-1

dsRNA-2

ORF I (1902 aa)

ORF II (1788 aa)

B

A

dsRNA-1dsRNA-2

Probe 1 Probe 2

Probe 3

Probe 2 Probe 1 Probe 3

1 kb

1067 1196

Proline-rich region

Estimated coding regionfor p85/80

Estimated coding regionfor p70


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LAKLVSS[82]LAKIVSS[82]LAKLVSS[82]***:***

SFHVGF[66]SFHVGF[66]SFHVGY[66]*****:

SN-GESRQLKFGRKTIFSTKKNSN-GESRQLKFGKKTIFSTKKNSN-GILREINFGGQKVRSTKKN** * *:::** :.: *****

[33]RTRIIFILPYE[119][33]RTRIIFILPYE[119][32]RTRAIFILPYQ[115]

*** ******:

AVASGEKQTKAANSIANLALI[18]AVASGEKQTKAANSIANLALI[18]ATASGEKQTKIMNSIANKALI[18]*.******** ***** ***

RVDGDDNYAVL[18]RYIYSRMNAK-----VKALVSTVG[56]RVDGDDNYAVL[18]RDTYSRMNAK-----VKALVSTVG[56]RVDGDDNYAIV[18]RSYYSEMNVK-----VKALASLTG[56]*********:: * **.**.* ****.* .*

FILTKIIFILTKIMFILTKIC******

QMTSVAITQMTSVAITQMTSIKIT****: **

SCMVSBMVLTSVCMVRYMV

BWYVCAYVPLVPEMV-1

MVEMGLC[36]LVRRGLC[36]LVRLGLC[36]LVKRGLC[36]LVEKGLC[36]

LVRNGLC[36]LVRRGLC[36]LVQEGLC[36]AVKDGLV[36] *: **

RMLFGA[14]KP-GMGLSVI-HQADAIFRDLRRMLFGP[14]KP-GMGLSLR-QQAKSLFDDLRRLLFGP[14]KP-GMGLSQK-WQFEALWKDLQRMLFGP[14]KP-GMGLSKA-SQVALLWEDLARMLFGP[14]KP-GMGLLTP-EQIRLVWDDVF

RVLFQN[14]KP-GFGLSTD-EQVLDFVESLARVLFQN[14]KP-GFGLSTD-EQVAEFMQILSRVLFQN[14]KP-GFGLSTD-TQTAEFLECLQRMLFRD[14]KP-GLGFSQD-HQVLAFTESVA*:** :: ** *:*:* * *. * : :

[15]FDWSVQDWELW[046][15]FDWSVQDWELW[046][15]FDWSVQKWELE[046][15]FDWSVQDWELW[046][15]FDWSVQDWELW[046]

[25]FDWSVADWMLH[047][25]FDWSVSDWLLE[046][26]FDWSVAYWMLE[047][25]FDWSVPMWLLE[047]

*****. *:*.

IMKSGSYCTSSTNSRIRCLMA[09]IAMGDDSVEGFIMKSGSYCTSSTNSRIRCLMA[09]IAMGDDSVEGWLMKSGSYCTSSTNSRIRCLMA[09]IAMGDDSVEGYLMKSGSYCTSSSNSRIRCLMA[09]IAMGDDSVEGWLMKSGSYCTSSTNSRIRCLMG[09]IAMGDDSVEGW

VQKSGSYNTSSSNSRIRVMAA[09]MAMGDDALESNVQKSGSYNTSSSNSRIRVMAA[09]MAMGDDALESAVQKSGSYNTSSSNSRIRVMAA[09]MAMGDDALEAPIQKSGSFNTSSTNSRMRYMLA[09]VTMGDDALESV:*****:****:***:* * * ::*******:

[19]PCATTPTGQL-----YAVEFCSHV[05]AFLTSWPKTLYRFLS[19]PCATTISGRL-----YEVEFCSHV[05]CWLASWPKTLFKYLS[19]PCAAEG-EIL-----KEVNFCSHS[05]CYLQSWAKTLFRYLE[19]ACPVLPNGDL-----KEVSFCSHL[05]AELETWPKCLFRYLS[19]PCKTR-GREL-----LEFNFCSHL[05]AELTSWPKALFRFLS

[07]KKLGFKVEVS-----GQLEFCSHI[06]ALPVNENKMIYKLIY[07]KSLGFKVEES-----SKLEFCSHI[06]AIPVNKAKMLYKLIH[07]KTLGFKVEVG-----RELEFCSHI[06]AVPVNTNKMLYKLIH[07]ARLGIKCERA-----EEFDFCSHL[06]VIPKNLEKMVYGLLS

:::****: .:* * **:* *:

ScV-L-A L--MNRG[60]ScV-L-BC L--ENGV[58]BfTV1 L--QGRF[60]HvV190S L--QGRY[61]MoV1 L--QGRG[60]Hm17V L--QGRM[62]GaVL1 L--LGRA[65]

UmVH1 L--YGRG[66]

WVPGGSIMPGGSWCVNGSWCVNGSWCVNGAWCVNGSWAVNGA

WLVSGS

[49]KY-EWG--KQ-RAIYGTDLRST[44][50]KY-EWG--KV-RALYGCDFSSH[43][45]KL-EAG--KD-RAIFACDTRSY[47][45]KL-ENG--KD-RAIFACDTRSY[47][50]KL-EHG--KT-RAIFACDTRSY[47][50]KL-EHG--KT-RAIFACDTQNY[47][48]KL-EAG--KT-RAIFACDTVNY[47]

[58]KLNETGG-KA-RAIYGVTLWHY[47]

FDYDDFNSQHS[052]YDFDDFNSQHS[052]LDYDNFNSHHS[045]LDYDNFNSQHS[045]LDYDDFNSQHS[045]LDYDDFNSHHA[046]LDYDDFNSHHS[046]

YDYPDFNSMHT[064]

TLLSGWRLTTFMNTVLTLFSGWRLTTFFNTALTLASGHRGTSFINSLLTLMSGHRATTFTNSVLTLMSGHRGTTFFNSILTMMSGHRGTMFINSVLTLMSGHRGTTFINSVL

GLYSGDRDTTLINTLL

NWAYM[14]VHNGDDVMISL[18]RAQPAKCNYCYL[12]LHNGDDVFAGI[18]RANTTKMNAAYI[14]LHAGDDVYIRA[18]RMNPTKQNAAYI[14]LHAGDDVYLRL[18]RMNPTKQNGVYI[14]MHTGDDVYMRL[18]RLNPTKQNAVYI[14]LHAGDDVYAVL[18]RMNSTKQNKAYL[14]LHVGDDVYFGV[18]RMNRMKQ

NIAYA[19]LCHGDDIITVH[18]KGQESKL

NL------FSISEFLRV[12]QYLSRSCATLVHSRINI------GTIAEFLRV[10]QYLTRGIATFTHSRVSIG-----FRNAEFLRL[08]GYVARTISTLVAGNWSIG-----YTGAEFLRL[08]GYLCRAIASLVSGSWSIG-----YKHAEFLRV[08]GYAARSIAALASGNWSVG-----VVCGEFLRM[08]GYVCRSIASFISGNWSVG-----HVSTEFLRN[08]GYFARAVASTVSGNW

MID-----HKHHEYLRI[09]GCLARCVATYVNGNW

ACDACV1 L--VGRR[73]CCRSCV L--VGRR[73]HvV145S L--LGRR[73]FoCV1 L--LGKE[73]PcV1 L--VGRG[73]

AbV1 L--TGRP[93] * *:

WLTKGSWLTKGSWMTKGSWLTKGSWLTKGG

SQVPGN*

[61]KLNECGY-[61]KLNECGY-[61]KLNENGH-[61]KLDENGH-[62]KY-EVGK-

[69]KH-EVGKN* * *

KD-RTLLPGSLFHY[44] FDKD-RTLLPGSLFHY[44] FDKD-RVLLPGGLLHY[44] YDKR-RALLPGSLMHY[43] FDKD-RTLLPGTLVHF[44] YD

AS-RSLWPAHLVHY[45] MD * * *. *.*: *

WANFNAFHSWANFNAFHSWANFNVQHSWANFNLYHSWADFNEQHS

YANFNEQHS:*:** **

[049]GLYSGWRGTSFLNSVLNSCYT[19]DHGGDDIDGGI[049]GLYSGWRGTSFLNSVLNSCYT[19]DHGGDDIDGGI[049]GLYSGWRGTTWDNTVLNGCYM[19]DQGGDDVDQEF[049]GLYSGWRGTTWINTVLNCVYT[19]DGGGDDHDGGL[049]GLYSGWRGTTWINTVLNFCYV[19]DHGGDDIDLGL

[053]GLLSGWRCTAYINNLINIAQY[19]DTGGDDGCADE ** **** *:: *.::* * ****

[18]EAQKIKQMIGI------DSEFFRI[08]GSATRALARFVSGNW[18]EAQKIKQMIGI------DSEFFRI[08]GSATRALARFVSGNW[18]EATKSKQMIGR------NSEFFRV[08]ASPVRGLATFVAGNW[18]KASAIKQMIAM------KSEFFRN[08]ASPVRTLTTFVNGKW[18]KANKWKQMFGT------RSEFFRN[08]ASPTRALASFVAGDW

[18]EFKDIKQLISS-----GTHEFFRL[08]GSVIRMLASAASGQW **::. **** .* * *: . *.*

LEV L--SGRA[73]PGV1 L--VGRA[79]RnBMV1 A--LGRF[72] **

LAPTGAVAPRGSFGSSGS... *:

[48]KV-ETG-LRS-RQIVPGPTKHW[51][48]KT-ESG-LRL-RQIIPGEIHQW[49][46]KR-EAG--KL-RQLLPGRIPHW[48]

* *:* : **::** :*

SDYADFNFLHTTDYADFNYLHTVDWADFNITHT *:**** **

[063]GLWSGWRTTSVI[064]SLWSGWRTTTMI[069]GLWSGWRTTSFI *****:.*

NNVFNEVYG[19]RRNGDDEDARA[18]DAQNNTMNLVYN[19]HVNGDDGDFEV[18]DVQNCSFNVAYC[19]VHAGDDFFGTY[18]EVN* :* .* *** :.:

PAKQKIGF-----TESEYTRV[08]NPIARGIASFASKQLLAA-----SHAEYLRI[08]GSIARSCASFAQKQLVGG-----EMGEFLRH[08]GSVMRSIGSF. ** :. .*: * ..: *. .**

TSSDLVGGDLVGSDL...**

Motif-I Motif-II Motif-III Motif-IV Motif-V Motif-VI Motif-VII Motif-VIII

Lute

o-v

irida

eS

obem

o-v

irus

Reo

-viri

dae

Totiv

irida

eC

hrys

oviri

dae

Una

ssig

ned

:*. *** *:** *. ** **** * :: *: ******.*** :**********:********. **********: ..**** . * :*.* *:::*.

SFV1 L--TGRS[91]CiTV1 S--FGRS[93]BpRV1 M--VGRP[67] **.

WIASGS[58]WMASGS[57]WMIKGS[59]*: .**

EKYENG--KA-RAIYGVEPMEKYENG--KA-RAIYGVAPMKGNEHG--KV-RAIYGSLYA: *:* *. *****

HY[49]LDYADFNRHHY[49]FDYADFNRHHY[47]LDYADFNAQ** :****** :

HT[053]GMFHT[053]GMFHS[055]GMF*: ***

SGTRSTDLINTLLNLAYF[20]VHQGDDVWVSN[18]SGTRSTDLLNTLLNLAYF[20]VHQGDDVWVSN[18]SGVRQTTLINTILNLTYH[20]YVLGDDGWVEF[18]**.*.* *:**:***:*. *** **.

IFQGSKQMFGP-----GRGEYLRV[08]GYFARSLANYLLRPIIFQKSKQMFGV-----GRGEYLRV[08]GYLGRAIANYILRPIEINAIKQLIGQ-----GKGEYLRL[09]GCPVRSLASFVHGNV :: **::* *:*****: * *::*.:: :

AfV L--VGRS[73]VcV L--IGRT[74]CnV1 L--VGRT[74]

WLTKGS[62]WLTKGS[63]WLTKGS[63]

KY-EVGN-KKY-ETGK-KKF-ETGR-K

D-RTLLPGTLVHF[44] YDD-RTLLPGSLIHF[44] YDD-RTLLPGTLIHF[44] YD

WANFNEQHS[049]WADFNEQHS[049]WADFNEQHT[049]

GLYSGWRGTTWVNTVLNFCYV[19]DHGGDDIVLGL[18]GLFSGWRGTTWINTVLNFCYS[19]DQGGDDIDSAL[18]GLYSGWRGTTWINSVLNFCYI[19]DHGGDDVDAAV[18]

KANKWKQMFGV------RSEFFRN[08]ASPTRALASFVAGDWNANAWKQMFST------RTEFFRN[08]ASPTRALASFCAGDWNANAWKQLFST------RSEFFRY[08]ASPTRALASFIAGDW

SaRV1 L--LGRA[61]WCVNGS[45]KL-EHG--KT-RAIFACDTRSY [47]LDFDDFNSHHS [045]TLPSGHRGTTIVNSVLNAAYI [14]LHTGDDVYIRA[18]RINPAKQSVG -----FGTGEFLRM[08]GYLARSVASFVSGNW

* . .*: * * * * **::. *: :*** *: : ** * * : *: ** * : ***: : : * : *:** * :: .

PGV2 L--PGPS[73]DSRV1 L--PGRN[73]FGV3 L--PGRS[72] * ** .

WAASGG[49]WAASGG[58]WAASGG[50]

******

SKFEKG--KR-RAIWNTAIEHY[54]PKFENG--KR-RIIWNTSLTHY[47]LKYEPG--KL-RSILNTSMEHY[47] *:* * * * * **:: **

WDFSDFNINHQ[064]WDYADFNINHS[062]WDFADFNINHT[063]**::******

CLASGERATSLASGERATSLQSGERAT.* ******

SFTNTILSRVYR[20]SQQGDDVFAAV[18]SFVNTVLSRAYR[20]DHQGDDVFATV[18]SWINTLRNHVDH[20]YKTGDDVFLTV[18]*: **: .:. : : ***** :*

AGQSYKVTLDY----APRGEFLRL[10]GYPIRAGLGLISGEFAGQLFKITCNH----TPSGEFLRQ[08]GYPIRSAMGLFSGEYAGQVSKIFVSYPELGGSRGEFVRY[10]GYPLRALTGLVHGEY*** *: .: . ***:* ***:*: **. **:

Meg

abirn

aviri

dae

**:* * .

.* . *

PRVARV HTV


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PcV

HvV145SFoCV1

CCRSCV ACDACV

AfV

VcV

CnV1

AbV1

GaVL1

HvV190SBfTV1 MoV1

Hm17V

SsRV1ScV-L-BC

ScV-L-A

UmVH1

RnBMV1

PGV1

LEV

BpRV1

SFV1

CiTV1

PGV2

DsRV1

FgV3

PRV

ARV

HTV

BWYV

CAYV

PLV

PEMV-1

SCMV

SBMV

LTSV

CMVRYMV

Chrysovirus

Rotavirus

Polerovirus

Sobem

oviru

s

Meg

abirn

aviru

s

Victorivirus

Totivirus

?

?

?

Enam

ovirus

1000

1000

1000

10001000

1000982

1000

1000

1000

1000

1000

1000

1000

1000 1000 1000

963

904

1000

0.1

704

706


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OnionBc-95 GarlicBc-38 GarlicBc-16 GarlicBc-72

Lesi

on L

engt

h (m

m)

Gro

wth

rate

(mm

/d)

V (dsRNA-) HV (dsRNA+)

Gar

licB

c-72

Gar

licB

c-16

A

B

C

0

6

12

18

24

0

4

8

12

16

n = 9

n = 5

aab

b

c

a abb

c


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A B

C D

VV

M

M

V

V

M

M


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0

5

10

15

20

25

0

2

4

6

8

10

12

14

Gro

wth

rate

(mm

/d)

Lesi

on le

ngth

(mm

)

a

b

cccd

deeefe-gf-hg-ig-ih-jh-jh-jh-kh-k

i-li-lj-lj-lj-mj-mj-mk-nk-n

k-nl-nl-nl-nm-om-om-ok-n

noopp

a

b

ccdcd

c-ec-e dedee

e e e e e e e e e e e e e e e e

e

e e e e e

e

e e e e

GP

72S

C30

GP

72S

C28

GP

72S

C18

GP

72S

C12

GP

72S

C09

GP

72S

C14

GP

72S

C08

GP

72S

C03

GP

72S

C13

GP

72S

C16

GP

72S

C22

GP

72S

C02

GP

72S

C31

GP

72S

C11

GP

72S

C01

GP

72S

C33

GP

72S

C06

GP

72S

C24

GP

72S

C04

GP

72S

C34

GP

72S

C21

GP

72S

C32

GP

72S

C15

GP

72S

C20

GP

72S

C35

GP

72S

C07

GP

72S

C05

GP

72S

C17

GP

72S

C10

GP

72S

C29

GP

72S

C25

GP

72S

C23

GP

72S

C27

GP

72S

C26

Gar

licB

c-16

Gar

licB

c-72

GP

72S

C19

dsRNA-1dsRNA-2

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

--

--

n = 9

n = 5

A

B


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A

B

0

2

4

6

8

10

12

GarlicB

c-38

38T

GP72SC35

72-35

-1

Gro

wth

rate

(mm

/d)

0

3

6

9

12

15

18

GarlicB

c-38

38T

GP72SC35

72-35

-1

Lesi

on le

ngth

(mm

)

****

****

dsRNA-dsRNA+

C dsRNA-dsRNA+

D


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Table 1. The origin of strains/isolates of Botrytis porri used in this study

Strain/isolate Origin (host, place and isolation time ) Pathogenicity Reference

GarlicBc-72 Allium sativum, Zhushan, China, 2008 Hypovirulent This study

OnionBc-95 Allium fistulosum, Qianjiang, China, 2008 Virulent Zhang et al., 2010

GarlicBc-38 Allium sativum, Laifeng, China, 2007 Virulent Zhang et al., 2010

GarlicBc-16 Allium sativum, Zhushan, China, 2007 Virulent Zhang et al., 2009

GP72SC35 A single-conidium isolate of GarlicBc-72,

2009

Virulent This study

72-35-1 GP72SC35 in a pairing-culture of

GP72SC35 and GarlicBc-72, 2009

Hypovirulent This study







72-38-1 GarlicBc-38-1 in a pairing-culture of

GarlicBc-38-1 and GarlicBc-72, 2009

Virulent This study



Virulent This study



Virulent This study

72-95-1 OnionBc-95 in a pairing-culture of

OnionBc-95 and GarlicBc-72, 2009

Virulent This study



Virulent This study



Virulent This study

38T GarlicBc-38-1 infected by BpRV1, 2010 Hypovirulent This study


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TABLE 2. Summary of the results of a BLASTP search for RdRp encoded by ORF I on dsRNA-1 of Botrytis porri RNA virus 1 (BpRV 1).

a See the legend for definitions of the virus name abbreviations in FIGURE 4.

Virus a RdRp

size (aa)

RdRp_4 conserved region BLASTP GenBank

Acc. No. Motif (I-VIII) length (aa) % Identity Overlap Bit score E value

Totivirus (Totiviridae)

UmVH1 1820 1142–1530 389 23 132/506 154 1e-34 NC_003823

ScV-L-A 731 169–520 352 19 67/254 73.2 4e-10 J04692

ScV-L-BC 863 306–654 349 20 98/424 67.0 3e-08 U01060

Chrysovirus (Chrysoviridae)

HvV145S 1086 426–805 380 23 164/616 121 1e-24 AF297176

PcV 1117 453–832 380 23 145/563 115 1e-22 AF296339

FoCV1 858 360–738 379 21 133/527 99.8 4e-18 EF152346

AfV 1114 451–829 379 21 135/561 97.1 3e-17 FN178512

CnV1 962 442–822 381 20 99/412 89.4 5e-15 GQ290650

ACDACV 1087 427–806 380 21 121/474 91.7 1e-15 NC_009947

VcV 1108 443–823 381 20 122/513 87.8 1e-14 HM004067

AbV1 1078 372–785 414 20 62/238 67.8 2e-08 X94361

CCRSCV 1087 427–806 380 21 64/282 60.5 3e-06 AJ781397

Victorivirus (Totiviridae)

Hm17V 845 243–594 352 21 115/469 87.0 3e-14 AB085814

SsRV1 838 241–589 349 22 74/256 79.0 8e-12 NC_001963

MoV1 845 227–575 353 20 98/407 78.2 1e-11 AB176964

GaVL1 825 221–573 353 20 103/427 68.6 1e-08 AF337175

BfTV1 838 242–585 344 21 57/219 67.4 3e-08 AM491608

HvV190S 835 241–585 345 21 64/241 59.7 5e-06 U41345

Megabirnavirus (Megabirnaviridae)

RnBMV1 1111 378–765 388 21 95/389 95.9 6e-17 AB512282

Unassigned

SFV1 1326 537–941 405 25 125/468 139 7e-30 GU979419

CiTV1 1338 550–953 404 23 145/591 108 6e-21 GU979420

PGV1 1414 669–1061 393 20 79/287 96.3 5e-17 AM111096

FgV3 1311 383–774 392 19 97/367 93.6 3e-16 NC_013469

DsRV1 1110 227–620 394 20 89/367 67.4 2e-08 NC_013699

PGV2 1153 230–624 395 18 87/374 66.2 5e-08 AM111097

LEV 1245 491–878 388 18 64/287 52.8 6e-04 AB429554


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Documents

Characterization of a Novel Bipartite Double-Stranded …jvi.asm.org/content/early/2012/04/06/JVI.00292-12.full.pdf33 encompass dsRNA-1 and dsRNA-2, and three stru ctural proteins