Arch Virol (2014) 159:3141–3144 DOI 10.1007/s00705-014-2156-6

ANNOTATED SEQUENCE RECORD

The complete nucleotide sequence of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-atrum M. Carmen Can˜izares • Encarnacio´n Pe´rez-Arte´s Marı´a D. Garcı´a-Pedrajas



Received: 10 March 2014 / Accepted: 19 June 2014 / Published online: 2 July 2014 Ó Springer-Verlag Wien 2014

Abstract We have characterized the bisegmented genome of a novel double-stranded RNA (dsRNA) virus isolated from the plant pathogenic fungus Verticillium alboatrum. We determined that its larger segment (dsRNA1) was 1747 base pairs in length and potentially encoded an RNA-dependent RNA polymerase of 539 amino acids, whereas the smaller segment (dsRNA2) was 1517 base pairs long and was predicted to encode a capsid protein of 435 amino acids. Homology searches and phylogenetic analysis confirmed that, as expected from its dsRNA banding profile, the identified virus was a new member of the family Partitiviridae, and we have therefore designated it V. albo-atrum partitivirus 1 (VaaPV1). This is the first report of a mycovirus identified in V. albo-atrum.

Introduction Mycoviruses are widespread in fungi, including phytopathogenic species [7, 12]. They are considered unusual in the sense that they lack an extracellular phase to their Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2156-6) contains supplementary material, which is available to authorized users. M. C. Can˜izares  M. D. Garcı´a-Pedrajas (&) Instituto de Hortofruticultura Subtropical y Mediterra´nea ‘‘La Mayora’’, Universidad de Ma´laga, Consejo Superior de Investigaciones Cientı´ficas (IHSM-UMA-CSIC), Estacio´n Experimental ‘‘La Mayora’’, 29750 Algarrobo-Costa, Ma´laga, Spain e-mail: [email protected] M. C. Can˜izares  E. Pe´rez-Arte´s Department of Crop Protection, Instituto de Agricultura Sostenible, IAS-CSIC, Alameda del Obispo s/n. Apdo 4084, 14080 Co´rdoba, Spain

replication cycle. Consequently, mycoviruses are not infectious in the classical sense; they are rather transmitted through intracellular mechanisms such as hyphal fusion or asexual sporulation [11]. Most mycoviruses have either double-stranded RNA (dsRNA) genomes or single-stranded RNA that produces dsRNA replicative intermediates [9]. Fungal cells lack large molecules of dsRNA. The isolation of these elements from fungi is used therefore as a tool for mycovirus diagnosis. The majority of known mycoviruses that form typical virus particles are classified based on the number and size of dsRNA genome segments into four families: Totiviridae, Chrysoviridae, Partitiviridae and Reoviridae. Members of the family Totiviridae have monopartite genomes, while the genomes of members of the Partitiviridae, Chrysoviridae and Reoviridae have two, four and eleven/twelve segments, respectively. The study of dsRNA profiles is commonly used to determine the incidence and variability of mycoviruses in fungal populations [12]. In recent years, mycoviruses from pathogenic species have received increasing attention since a number of them have been shown to induce hypovirulence in the fungal host and might therefore be exploited as biological control agents. Verticillium albo-atrum and V. dahliae are the two major phytopathogenic species within the cosmopolitan genus Verticillium. They affect a wide variety of crops, and their different geographic distribution is determined by temperature, with V. dahliae appearing to be favored by higher temperatures than is V. albo-atrum [5]. Although dsRNAs have been detected in all major taxonomic groups of filamentous fungi [7], in the case of Verticillium spp., reports on the molecular characterization of mycoviruses has been restricted to a chrysovirus and a partitivirus, both of which were found in V. dahliae infecting cotton in China [3, 4]. In the study presented below, two dsRNA segments

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were isolated from V. albo-atrum, the other major causal agent of Verticillium wilt, and fully sequenced. Homology searches and phylogenetic analysis showed that these dsRNAs constitute the genome of a partitivirus, which we have designated V. albo-atrum partitivirus 1 (VaaPV1), and which is taxonomically distinct from the aforementioned V. dahliae partitivirus.

Provenance of the virus material The mycovirus characterized in this work was obtained from V. albo-atrum isolate 383-2. This is a monoconidial isolate originally obtained by G. Lazarovits (AAFC, London, Ontario) from potato grown in Ontario, Canada, in 1986. It was provided to us by K. F. Dobinson (AAFC, London, Ontario), who had maintained it in her collection in long-term storage as a filter stock at -20 °C. Upon reception in 2009, we maintained it at -20 °C as a glycerol stock until use. To investigate the presence of mycovirus(es), in V. alboatrum 383-2, the fungus was grown for 4 days in PDB

(a)

(potato dextrose broth), and dsRNAs were extracted from the mycelia using CF-11 cellulose [9] and electrophoretically fractionated on a 1 % agarose gel. Two dsRNA bands of approximately 1.7 and 1.5 kb (dsRNA1 and dsRNA2) were detected (Fig. 1a). This dsRNA banding pattern clearly corresponded to that of members of the family Partitiviridae, whose genomes are composed of two dsRNA segments of sizes ranging between 1.4 and 2.4 kb [6]. Both dsRNAs were purified individually and subjected to reverse transcription (RT) and PCR amplification using random hexamer priming (Invitrogen, Carlsbad, CA, USA) to produce a set of cDNA clones for each segment. Analysis of these clones generated partial sequences of both RNAs. RT-PCR amplification using sequence-specific primers was then used to produce cDNA clones for dsRNA regions not covered by the cDNA library clones. Finally, clones carrying the terminal sequences of the dsRNAs were generated by T4 RNA ligase oligonucleotide-mediated amplification as described by Xie et al. [14]. The PCR primers used are listed in Supplementary Table 1. The fulllength cDNA sequence for dsRNA 1 and dsRNA2 were obtained by assembling of the partial sequences in the

(b)

(c)

Fig. 1 Characterization of V. albo-atrum partitivirus 1 (VaaPV1). (a) dsRNA banding pattern obtained from V. albo-atrum strain 383-2 analyzed by agarose gel electrophoresis. The size standard is shown on the left. (b) Nucleotide sequence alignment of the 5’-UTR of the two genomic segments of Verticillium albo-atrum partitivirus 1 (VaaPV1). Black shading indicates identical nucleotides in the two segments. (c) Alignment of amino acid sequences of the RdRp motifs of VaaPV1 and those of selected viruses in the former genus Partitivirus. Conserved amino acids, motifs 3–8, are showed in horizontal lines above the amino acid areas. Asteriks indicate identical amino acid residues (shaded), and colons show similar

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residues. Numbers in brackets correspond to the number of amino acid residues separating the motifs. The abbreviations of the virus names are as follows: AoV1, Aspergillus ochraceous virus 1; DdVl, Discula destructiva virus 1; DdV2, D. destructiva virus 2; FsV1, Fusarium solani virus 1; GaRV-MS1, Gremmeniella abietina virus MS1; GaRV-MS2, G. abietina virus MS2; OPV1, Ophiostoma partitivirus 1; PsV-F, Penicillium stoloniferum virus F; PsV-S, P. stoloniferum virus S; VdPV1, V. dahliae partitivirus 1; BfPV1, Botryotinia fuckeliana partitivirus 1; AfuPV1, Aspergillus fumigatus partitivirus 1; UvPV1, Ustilaginoidea virens partitivirus 1; VaaPV1, V. albo-atrum partitivirus 1

Novel partitivirus from Verticillium albo-atrum

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different cDNA clones (see Supplementary Fig. 1). All cDNA clones were sequenced in both orientations, and all sequences were confirmed by using at least two independent overlapping clones. Sequencing was performed at the Genomic Unit of the University of Co´rdoba, Spain. Nucleotide sequences were edited and translated using BIOEDIT [8]. BLASTP was used for homology database searching [1], and MEGA 5 for sequence alignment (using CLUSTAL W) and phylogenetic tree construction [13]. On

Sequence properties We determined that the complete dsRNA1 sequence was 1747 base pairs (bp) in length, and putatively encoded a

(b)

AfuPV1 UvPV1 71

AfuPV1

VdPV1

100

UvPV1

BfPV1

VdPV1

74

100

Partitivirus (II)

DdV1

100

DdV2 OPV1 AoV1

100 83

PsV-S GaRV-MS1

100 100

DdV1

99

DdV2 BfPV1 OPV1

100

AoV1

99

PsV-S

GaRV-MS2

100

92

GaRV-MS1

FsV1 94

100

VaaPV1 87 79

HetRV3

HetRV3 86

FvBV CrV1 CpV AhV-2H

93

HaV 100

HetRV2

100

RhsV-717

FvBV PsV-F

Partitivirus (I)

100 100 100

VaaPV1

HmV

100

RhsV-717

100

RnV1-W8 PoV1

100 100

100

HetRV8 FpV1 HetRV2

RnV1-W8

100

PoV1

100

AhV-2H

70

FpV1

83 100

GaRV-MS2 FsV1

PsV-F 100

Partitivirus (II)

(a)

the basis of the aligned amino acid (aa) sequences, an unrooted tree was created using the neighbor-joining method.

100

HetRV8

0.1

Fig. 2 Phylogenetic analysis of VaaPV1 and other members of the former genus Partitivirus. (a) Phylogenetic tree based on the RdRp amino acid sequences. (b) Phylogenetic tree based on the CP amino acid sequences. Phylogenetic trees were constructed using the program MEGA 5.0, and generated by the neighbor-joining method. Bootstrap scores (1000 replicates) are shown at nodes when they are higher than 70 %. Vertical grey bars on the right indicate coincidence with the reported grouping of partitiviruses, i.e., Partitivirus I and II. Sequences of the RdRp and CP genes were obtained from the GenBank database for the following viruses (abbreviations and accession numbers are in parentheses): V. albo-atrum partitivirus 1 (VaaPV1; JK476945, KJ476946), Aspergillus fumigatus partitivirus 1 (AfPV1; FN376847, FN398100), Aspergillus ochraceous virus 1 (AoV1; EU118277, EU118278), Atkinsonella hypoxylon virus (AhV2H; L39125, L39126), Botryotinia fuckeliana partitivirus 1 (BfPV1; AM491609. AM491610), Ceratocystis polonica virus (CpV; AY260756, AY247205), Ceratocystis resinifera virus 1 (CrV1; AY603051, AY603052), Discula destructiva virus 1 (DdV1; NC_002797, NC_002800), D. destructiva virus 2 (DdV2;

Crv1

100

CpV

0.1

NC_003710, NC_003711), Flammulina velutipes browning virus (FvBv; AB465308, AB465309), Fusarium poae virus 1 (FpV1; NC_003884, NC_003883), Fusarium solani virus 1 (FsV1; D55668, D55669), Gremmeniella abietina virus MS1 (GaRV-MS1; NC_004018, NC_004019), G. abietina virus MS2 (GaRV-MS2; NC_006444, NC_006445), Heterobasidium RNA virus 2 (HetRV2; HM565953, HM565954), Heterobasidium RNA virus 3 (HetRV3; FJ816271, FJ816272), Heterobasidium RNA virus 8 (HetRV8; JX625227, JX625228.1), Helicobasidium mompa partitivirus (HmV; AB110979), Heterobasidium annosum partitivirus (HaPV; AF473549), Ophiostoma partitivirus 1 (OPV1; AM087202, AM 087203); Penicillium stoloniferum virus F (PsV-F; AY7386, AY738337), P. stoloniferum virus S (PsV-S; AY156521, AY156522), Pleorotus ostreatus virus (PoV; AY533036, AY533038), Rhizoctonia solani virus 717 (RhsV-717; AF33290, AF133291), Rosellinia necatrix virus 1-W8 (RnV1-W8; AB113347, AB113348), Ustilaginoidea virens partitivirus 1 (UvPV1; KC503898, KC503899), V. dahliae partitivirus 1 (VdPV1; KC422244.1; KC422243.1)

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539-aa protein, whereas the dsRNA2 was 1517 bp long, and potentially encoded a protein of 435 aa (GenBank database accession numbers KJ476945 and KJ476946, respectively). dsRNA1 and 2 contained both 53 nt 5’-untranslated regions (UTRs). The sequences of the two 5’-UTRs were *66 % identical, and they contained a 5’-terminal identical stretch of 11 nts (Fig. 1b). The 3’-UTRs differed in length, 74 nt for RNA1 and 156 nt for RNA2, and were apparently not conserved. The predicted protein encoded by dsRNA1 contained the conserved motifs characteristic of the RNA-dependent RNA polymerases (RdRps) of mycoviruses (Fig. 1c) [2]. A homology search with BLASTP indicated that this protein was closely related to the RdRps of viruses in the family Partitiviridae. In particular, the most closely related RdRp, showing 64 % aa sequence identity, was that of Fusarium solani virus 1 (FsV1) [10], while the sequence identity with the RdRp of the partitivirus reported in the genus Verticillium, V. dahliae VdPV1 [4], was 61 %. As described in the previous taxonomy, the former genus Partitivirus comprised two major branches [6], and a comprehensive phylogenetic analysis of RdRp aa sequences placed VaaPV1, as expected, in the branch that contained most of the partitiviruses described in ascomycetous fungi (Fig. 2a), where VaaPV1 and FsV1 clustered together. The putative coat protein (CP) encoded by dsRNA2 shares *40 % aa sequence identity with CPs of partitiviruses that infect ascomycetous fungi. A phylogenetic tree based on the full-length sequences of these CPs also placed VaaPV1 in the Ascomycota branch of the partitiviruses, and clustering together with FsV1, which confirms that VaaPV1 has a closer relationship to FsV1 than to V. dahliae VdPV1 (Fig. 2b). In summary, the banding profile, sequence similarities, and the high bootstrap values observed in the phylogenetic analysis, support the classification of the virus isolated from V. albo-atrum strain 383-2 as a novel unreported member of the family Partitiviridae. As far as we know, this is the first report of a mycovirus identified in the plant pathogenic fungus V. albo-atrum. Acknowledgements This work was supported by the Spanish Ministry of Science and Innovation Grant No. AGL2009-13445

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awarded to M.D.G.-P. We thank K.F.D. and AAFC for the V. alboatrum strain used in this study.

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The complete nucleotide sequence of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-atrum.

We have characterized the bisegmented genome of a novel double-stranded RNA (dsRNA) virus isolated from the plant pathogenic fungus Verticillium albo-...
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