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Characterization of a novel Alternavirus infecting the fungal pathogen Fusarium solani
Abstract
A novel dsRNA mycovirus was found in Fusarium solani (F. solani) strain NW-FVA 2572. The fungus was originally isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L. The viral genome is composed of four segments, which range from around 3.5 kbp to 1.7 kbp (RNA 1: 3522 bp; RNA 2: 2633 bp; RNA 3: 2403 bp; RNA 4: 1721 bp). The segments share a conserved and capped 5’-terminus and their 3’-termini are polyadenylated. Protein sequencing showed that the viral RdRP is encoded on segment 1. The virus clusters together with Aspergillus mycovirus 341 (AsV341), Aspergillus heteromorphus alternavirus 1 (AheAV1), Aspergillus foetidus virus-fast (AfV-F) and Cordyceps chanhua alternavirus 1 (CcAV1). As highest value, the RdRP showed 61.50 % identical amino acids with P1 of the AfV-F. The capsid protein is encoded on segment 3, the proteins encoded on RNA 2 and RNA 4 are of unknown function. Segment 4 harbors large UTRs (186 nts at the 5’-terminus and 311 nts at the 3’-terminus).
Based on its genome organization and phylogenetic position, the virus is suggested to be a new member of the proposed family Alternaviridae and was therefore named Fusarium solani alternavirus 1 (FsAV1). This is the first report of an Alternavirus infecting a fungus of the F. solani species complex (FSSC).
... FoAV1, an alternavirus, was identified in F. oxysporum isolated from diseased stems of L. brownii with the typical symptoms of Fusarium wilt [11]. Besides FoAV1, there were seven other alternaviruses determined in seven species of Fusarium, respectively [11,[13][14][15][16][17][18][19]. However, to the best of our knowledge, this is the first report of an alternavirus infecting FOM and causing muskmelon Fusarium wilt. ...
... proposals/approved?fid=11959#block-teamplus-page-title, accessed on 5 March 2024). Thus far, eighteen mycoviruses have been assigned as members of the family Alternaviridae [11,[13][14][15][16][17][18][19][47][48][49][50][51][52][53][54][55]. Except for FaAV1, which is composed of two dsRNA segments [13], the remaining seventeen alternaviruses contain three or four dsRNA segments [11,[14][15][16][17][18][19][47][48][49][50][51][52][53][54][55]. ...
... Thus far, eighteen mycoviruses have been assigned as members of the family Alternaviridae [11,[13][14][15][16][17][18][19][47][48][49][50][51][52][53][54][55]. Except for FaAV1, which is composed of two dsRNA segments [13], the remaining seventeen alternaviruses contain three or four dsRNA segments [11,[14][15][16][17][18][19][47][48][49][50][51][52][53][54][55]. Among them, eleven alternaviruses, namely Aspergillus heteromorphus alternavirus 1 (AheAV1) [49], Cordyceps chanhua alternavirus 1 (CcAV1) [52], Dactylonectria torresensis alternavirus 1 (DtAV1) [53], FgAV1 [14], FiAV1 [15], FnAV1 [16], FpgAV1 [18], FpAV1 [17], Ilyonectria crassa alternavirus 1 (IcAV1) [51], Ilyonectria robusta alternavirus 1 (IrAV1) [53], and Suillus luteus alternavirus 1 (SluAV1) [55], contain three dsRNA segments, while six alternaviruses, including AaV1 [47], Aspergillus foetidus virus (AfV) [54], Aspergillus virus 341 (AsV341) [50], FoAV1 [11], FsAV1 [19], and Stemphylium lycopersici alternavirus 1 (SlAV1) [48], contain four dsRNA segments. ...
In the current study, a novel strain of Fusarium oxysporum alternavirus 1 (FoAV1) was identified from the Fusarium oxysporum f. sp. melonis (FOM) strain T-BJ17 and was designated as Fusarium oxysporum alternavirus 1-FOM (FoAV1-FOM). Its genome consists of four dsRNA segments of 3515 bp (dsRNA1), 2663 bp (dsRNA2), 2368 bp (dsRNA3), and 1776 bp (dsRNA4) in length. Open reading frame 1 (ORF1) in dsRNA1 was found to encode a putative RNA-dependent RNA polymerase (RdRp), whose amino acid sequence was 99.02% identical to that of its counterpart in FoAV1; while ORF2 in dsRNA2, ORF3 in dsRNA3, and ORF4 in dsRNA4 were all found to encode hypothetical proteins. Strain T-BJ17-VF, which was verified to FoAV1-FOM-free, was obtained using single-hyphal-tip culture combined with high-temperature treatment to eliminate FoAV1-FOM from strain T-BJ17. The colony growth rate, ability to produce spores, and virulence of strain T-BJ17 were significantly lower than those of T-BJ17-VF, while the dry weight of the mycelial biomass and the sensitivity to difenoconazole and pydiflumetofen of strain T-BJ17 were greater than those of T-BJ17-VF. FoAV1-FOM was capable of 100% vertical transmission via spores. To our knowledge, this is the first time that an alternavirus has infected FOM, and this is the first report of hypovirulence and increased sensitivity to difenoconazole and pydiflumetofen induced by FoAV1-FOM infection in FOM.
... The finding of a novel virus in Aspergillus foetidus (AfV-F) that was phylogenetically related to AaV1 led to the proposal of the new family "Alternaviridae" with AaV1 as its type member [3]. Members of this proposed family possess a genome consisting of three to four monocistronic dsRNA segments, which range from 1.4 kbp (dsRNA 4) to 3.6 kbp (dsRNA 1) [3][4][5][6][7][8][9][10][11], and a polyA tail at the 3' end. Wu et al. [12] and Lutz et al. [10] experimentally showed that the 5' ends of the AaV1 and of Fusarium solani alternavirus 1 (FsAV1) genome segments are capped. ...
... Members of this proposed family possess a genome consisting of three to four monocistronic dsRNA segments, which range from 1.4 kbp (dsRNA 4) to 3.6 kbp (dsRNA 1) [3][4][5][6][7][8][9][10][11], and a polyA tail at the 3' end. Wu et al. [12] and Lutz et al. [10] experimentally showed that the 5' ends of the AaV1 and of Fusarium solani alternavirus 1 (FsAV1) genome segments are capped. It is not known if other members of this family are also capped at their 5' end. ...
... It is not known if other members of this family are also capped at their 5' end. It was also shown that the proteins encoded on segment 1 and on segment 3 are structural and were proposed to represent the RdRPs and the capsid proteins, respectively [4,10,12]. The proteins encoded on dsRNA 2 and dsRNA 4 are suggested to be non-structural and are of unknown function. ...
A novel dsRNA mycovirus named Ilyonectria crassa alternavirus 1 (IcAV1) was found in Ilyonectria crassa isolate NW-FVA
1829. The fungus was isolated from an ash (Fraxinus excelsior L.) necrotic trunk disc infected with Hymenoscyphus fraxineus
[(T. Kowalski) Baral, Queloz, Hosoya] causing ash dieback. The complete genome of IcAV1 is composed of three segments,
each containing a single ORF on the positive-sense RNA. The extreme 5’ UTRs of dsRNA 1 (3604 bp), dsRNA 2 (2547 bp),
and dsRNA 3 (2518 bp) share a conserved hexadecamer sequence (5’-GGC TGT GTG TTT AGTT-3’) and are capped. The
3’ UTRs are polyadenylated. In silico analysis showed that the viral RdRP is encoded on dsRNA 1 and the capsid-protein
subunits are encoded on dsRNA 3. Maximum-likelihood analysis of the aa sequence of the viral RdRP showed that IcAV1
clusters with alternaviruses from Fusarium spp., while the type member of the proposed family "Alternaviridae", Alternaria
alternata virus 1 (AaV1), formed a clade together with Stemphylium lycopersici mycovirus (SlV). The function of the protein
encoded on segment 2 is unknown. Based on its genome organization and its phylogenetic position, IcAV1 is suggested to
be a new member of the proposed family "Alternaviridae". This is the first report of a mycovirus infecting I. crassa.
... At least forty-seven mycoviruses and their full-length genomes have been reported in eighteen species of Fusarium [27,52]; however, no mycoviruses have been identified in F. avenaceum, one of the main phytopathogenic species of Fusarium causing potato dry rot. In the present study, FaAV1 was extracted from F. avenaceum strain GS-WW-224 and identified to be a new alternavirus, which is the first time that an alternavirus has been identified in F. avenaceum. ...
... In the present study, FaAV1 was extracted from F. avenaceum strain GS-WW-224 and identified to be a new alternavirus, which is the first time that an alternavirus has been identified in F. avenaceum. Thus far, eleven mycoviruses have been designated as members of the proposed family Alternaviridae [7,[19][20][21][22][23][24][25][26][27][28][29]. Among them, FiAV1 [7], FpAV1 [23], FgAV1 [24], AheAV1 [25], and CcAV1 [28] contain three dsRNA segments, while AsV341 [19], AaV1 [20,21], AfV [22], FoAV1 [26], FsAV1 [27], and SlAV1 [29] contain four dsRNA segments. ...
... Thus far, eleven mycoviruses have been designated as members of the proposed family Alternaviridae [7,[19][20][21][22][23][24][25][26][27][28][29]. Among them, FiAV1 [7], FpAV1 [23], FgAV1 [24], AheAV1 [25], and CcAV1 [28] contain three dsRNA segments, while AsV341 [19], AaV1 [20,21], AfV [22], FoAV1 [26], FsAV1 [27], and SlAV1 [29] contain four dsRNA segments. The genomic composition of FaAV1 identified in the present study, however, only contains two dsRNA segments. ...
A novel virus with a double-stranded RNA (dsRNA) genome was isolated from Fusarium avenaceum strain GS-WW-224, the causal agent of potato dry rot. The virus has been designated as Fusarium avenaceum alternavirus 1 (FaAV1). Its genome consists of two dsRNA segments, 3538 bp (dsRNA1) and 2477 bp (dsRNA2) in length, encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein (HP), respectively. The virions of FaAV1 are isometric spherical and approximately 30 nm in diameter. Multiple sequence alignments and phylogenetic analyses based on the amino acid sequences of RdRp and HP indicated that FaAV1 appears to be a new member of the proposed family Alternaviridae. No significant differences in colony morphology and spore production were observed between strains GS-WW-224 and GS-WW-224-VF, the latter strain being one in which FaAV1 was eliminated from strain GS-WW-224. Notably, however, the dry weight of mycelial biomass of GS-WW-224 was higher than that of mycelial biomass of GS-WW-224-VF. The depth and the width of lesions on potato tubers caused by GS-WW-224 were significantly greater, relative to GS-WW-224-VF, suggesting that FaAV1 confers hypervirulence to its host, F. avenaceum. Moreover, FaAV1 was successfully transmitted horizontally from GS-WW-224 to ten other species of Fusarium, and purified virions of FaAV1 were capable of transfecting wounded hyphae of the ten species of Fusarium. This is the first report of an alternavirus infecting F. avenaceum and conferring hypervirulence.
... According to the 9 th report on subviral agents by the ICTV [66], satellite-like nucleic acids are defined to be distinct from their helper virus and are either coding for no or for a non-structural protein. As it was shown for an additional segment (dsRNA 4) for the Fusarium solani alternavirus 1 (FsAV1) [67], the third segment of DfPV1 also has extended NTRs and is therefore distinct from segment 1 and 2. The criterium of encoding either no protein or a non-structural protein is fulfilled since protein patterns of VLPs did not show any band with the expected size of about 30 kDa. Several related proteins to P3 were found by BLASTp which are the corresponding proteins of other tripartite gammapartitiviruses. ...
... In contrast, the genus Aspergillus belongs to the class Eurotiomyces and is not related to the genus Botryosphaeria which is accommodated in the class Dothideomycetes. Therefore, the third segment of AoV may have been acquired independently by horizontal gene transfer (HGT) as it was speculated by Wang et al. [68] for a papain-like protease domain on dsRNA 2 of Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), for a Phytoreo_S7 domain in non-phytoreoviruses by Liu et al. [24] and from Lutz et al. [67] for segment 4 of FsAV1. ...
Two novel dsRNA mycoviruses were found in different isolates of Diplodia fraxini, NW FVA 1581 and NW FVA 1706, which were isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L. Both mycelia are infected by a novel fusagravirus, which was named Diplodia fraxini fusagravirus 1 (DfFV1), and isolate NW FVA 1706 is additionally infected by a novel partitivirus, which was denominated as Diplodia fraxini partitivirus 1 (DfPV1). The one-segmented, bicistronic genome of DfFV1 is composed of about 8,500 bp. Their ORFs are connected by a 1 slippery heptamer sequence and the 3’ terminal ORF is coding for the viral RdRp. The genome of DfPV1 is composed of three, monocistronic dsRNA segments ranging from 1,755 bp (dsRNA 1) over 1,588 bp (dsRNA 2) to 1,233 bp (dsRNA 3). Based on genome organization and phylogenetic positions, DfFV1 was assigned to the proposed family of “Fusagraviridae” and DfPV1 to the genus Gammapartitivirus within the family of Partitiviridae. Ultra-structural analysis showed that polysomal structures were stabilized in the single infection and none of these structures could be isolated in the double infection. It is assumed that DfFV1 has an opportunistic lifestyle, being either protected by ribosomes or by transcapsidation from particles of DfPV1.
... The presence of dsRNA in Aspergillus species was initially reported in 1970 (Banks et al. 1970), and subsequent screenings of large Aspergillus strain panels confirmed the existence of various dsRNA elements (Kotta-Loizou and Coutts 2017). Mycoviruses such as chrysovirus (Takahashi-Nakaguchi et al. 2020), partitivirus (Jiang et al. 2021), totivirus (Jiang et al. 2022), polymycovirus (Zoll et al. 2018), and viruses in proposed families Alternaviridae (Lutz et al. 2022) and Yadokariviridae (Kotta-Loizou and Coutts 2017) have been detected in various Aspergillus species with incidences ranging from 7 to 50% (Kotta-Loizou and Coutts 2017). Notably, however, no sequence data of viruses from A. ochraceopetaliformis have been published. ...
An airborne microflora isolate, Aspergillus ochraceopetaliformis RCEF7483, was found to harbor seven dsRNA elements, indicating co-infection with a novel chrysovirus and a known partitivirus. Sequence analysis and RT-PCR confirmed dsRNA5–7 as components of Aspergillus ochraceous virus (AOV), a member of the Partitiviridae family. In light of its distinct host, we have designated it Aspergillus ochraceopetaliformis partitivirus 1 (AoPV1). The dsRNA segments, named dsRNA1–4, with lengths of 3706 bp, 3410 bp, 3190 bp, and 3158 bp, respectively, constitute the genome of a novel chrysovirus designated Aspergillus ochraceopetaliformis chrysovirus 1 (AoCV1). The dsRNA1–4 segments contain five open-reading frames (ORF1–5). Specifically, ORF1 encodes a putative RNA-dependent RNA polymerase (RdRp) with a length of 1112 amino acids, and ORF2 encodes a putative coat protein (CP) spanning 976 amino acids. Additionally, ORF3–5 encode hypothetical proteins (HP1, HP2, and HP3) with lengths of 108, 843, and 914 amino acids, respectively. Comparative analysis revealed the highest similarity of dsRNA1–4 with corresponding proteins in Aspergillus terreus chrysovirus 1 (AtCV1) (RdRp, 66.58%; CP, 51.02%; HP2, 61.80%; and HP3, 41.30%). Due to falling below the threshold for a new species in the Chrysoviridae, we propose that dsRNA1–4 in A. ochraceopetaliformis strain RCEF7483 constitute the novel chrysovirus AoCV1. Moreover, phylogenetic analysis using RdRp amino acid sequences placed AoCV1 within the Alphachrysovirus genus of the Chrysoviridae family, clustering with AtCV1 and other alphachrysoviruses. Our study contributes to the understanding of mycoviruses in A. ochraceopetaliformis and expands our knowledge of the diversity and evolution of chrysoviruses in fungal hosts.
... To date, Alternaviridae has exclusively been discovered in Ascomycetes, with eight alternaviruses identi ed in Fusarium species. These include Fusarium incarnatum alternavirus 1 (FgAV1) [7], Fusarium incarnatum alternavirus 1 (FiAV1) [8], Fusarium oxysporum alternavirus 1 (FoAV1) [9], Fusarium poae alternavirus 1 (FpAV1) [10], Fusarium poae alternavirus 2 (FpAV2) [10], Fusarium solani alternavirus 1 (FsAV1) [11], Fusarium nanum alternavirus 1 (FnAV1) [12], and Fusarium avenaceum alternavirus 1 (FaAV1) [13]. Among these viruses, FoAV1 has been shown to signi cantly reduce the virulence of its host [9], while FaAV1, conversely, signi cantly enhances host virulence [13]. ...
Three dsRNA sequences were detected from the Fusarium pseudograminearum strain CF14029, a pathogen causing Fusarium Crown Rot in China. Characterization and sequencing analyses confirmed that these dsRNA sequences originated from the same virus. The virus consists of three dsRNA segments: dsRNA1 (3,560 nt in length) encoding an RNA-dependent RNA polymerase (RdRp), dsRNA2 (2,544 nt in length), and dsRNA3 (2,478 nt in length) which encodes a coat protein (CP). Phylogenetic analysis based on the RdRp and CP amino acid sequences revealed a high similarity of this virus with alternaviruses isolated from other Fusarium fungi of the genus Alternavirus within the family Alternaviridae . As a novel member of the Alternavirus genus, this virus is provisionally named Fusarium pseudograminearum alternavirus 1 (FpgAV1). Similar to other alternaviruses in Fusarium fungi, the positive-sense strand of each genomic dsRNA of FpgAV1 possesses a poly(A) tail and a distinctive 5'-terminal nonamer sequence (5’-GGC TGT GTG-3’). This study represents the first comprehensive description of the genomic sequence of an Alternavirus identified in F. pseudograminearum .
... Some species not only reduce yield production and value of cereals but produces toxins in crops which can disturb the health of animal and human. Now, mycoviruses have been reported in Fusarium graminearum, F. solani, F. oxysporum, F. poae, F. virguliforme and F. boothii [80,83,[87][88][89][90]. Fusarium viruses belong to Totiviridae, Partitiviridae and Chrysoviridae [91]. ...
All key families of plant-infecting fungi have been observed to be infected by mycoviruses, which are substantial and ubiquitous viruses. Mycoviruses, like all other plant and animal viruses, must reproduce in live cells. These viruses spread either during spore production or cell division. Few mycoviruses are known to have a positive single-stranded RNA genome, while the majority have double-stranded RNA genomes. Few mycoviruses have been identified as the causative agents of triggering atypical pigmentation and sporadic growth to alter the sexual reproduction of the host. These viruses are categorized into taxonomic groups as developed from plant viruses. mycoviruses are notable due to their amazing capacity to cause a dramatic decrease in the virulence (hypovirulence) of the host. It has been observed that mycoviruses do not necessarily cause hypovirulence, but they also impart hypervirulence to the host fungi. The current effort summarizes the data regarding mycoviral diversity, taxonomy and their role in the pathogenesis of pathogenic fungi.
... Quadriviruses were originally discovered in Rosellinia necatrix (Lin et al., 2012), and later in, e.g., B. cinerea (Ruiz-Padilla et al., 2021), Leptosphaeria biglobosa and Thelonectria (Lutz et al., 2022a). In turn, family "Alternaviridae" was named after Alternaria alternata, which was among the first hosts identified (Aoki et al., 2009) and is also found in Cordyceps chanhua, different species of Fusarium, and Stemphylium lycopersici (Lutz et al., 2022b). Both families have been detected so far exclusively in fungi. ...
Knowledge of mycovirus diversity, evolution, horizontal gene transfer and shared ancestry with viruses infecting distantly related hosts, such as plants and arthropods, has increased vastly during the last few years due to advances in the high throughput sequencing methodologies. This also has enabled the discovery of novel mycoviruses with previously unknown genome types, mainly new positive and negative single-stranded RNA mycoviruses ((+) ssRNA and (−) ssRNA) and single-stranded DNA mycoviruses (ssDNA), and has increased our knowledge of double-stranded RNA mycoviruses (dsRNA), which in the past were thought to be the most common viruses infecting fungi. Fungi and oomycetes (Stramenopila) share similar lifestyles and also have similar viromes. Hypothesis about the origin and cross-kingdom transmission events of viruses have been raised and are supported by phylogenetic analysis and by the discovery of natural exchange of viruses between different hosts during virus-fungus coinfection in planta. In this review we make a compilation of the current information on the genome organization, diversity and taxonomy of mycoviruses, discussing their possible origins. Our focus is in recent findings suggesting the expansion of the host range of many viral taxa previously considered to be exclusively fungal, but we also address factors affecting virus transmissibility and coexistence in single fungal or oomycete isolates, as well as the development of synthetic mycoviruses and their use in investigating mycovirus replication cycles and pathogenicity.
... Up to now, eleven viruses have been assigned to the proposed family Alternaviridae. The eponymous alternaria alternata virus 1 (AaV-1) was identified in 2009 and the previously found aspergillus mycovirus 341 (AMV) was assigned to the family of which nine more viruses were discovered all around the world in the following years (Aoki et al. 2009;Gilbert et al. 2019;Hammond et al. 2008;He et al. 2018;Kozlakidis et al. 2013;Osaki et al. 2016;Wen et al. 2021;Zhang et al. 2019Zhang et al. , 2022Lutz et al. 2022). One of the most prominent features of this family is the ADD sequence triplet (motif VI) in the RdRp amino acid (aa) sequence. ...
Since the first report in 2009, at least ten additional viruses have been identified and assigned to the proposed virus family Alternaviridae. Here we report two new mycoviruses tentatively assigned to this family, both identified as members of the fungal family Nectriaceae, which were isolated from surface-disinfected apple roots (Malus x domestica, Borkh.) affected by apple replant disease (ARD). ARD is a highly complex, worldwide-occurring disease resulting from plant reactions to a disturbed (micro)-biome and leads to high economic losses every year. The first alternavirus characterized in this study was identified in a Dactylonectria torresensis isolate. The virus was tentatively named dactylonectria torresensis alternavirus 1 (DtAV1) as the first member of the proposed new species Alternavirus dactylonectriae. The second virus was identified in an isolate of Ilyonectria robusta and was tentatively named ilyonectria robusta alternavirus 1 (IrAV1) as the first member of the proposed new species Alternavirus ilyonectriae. Full genomic sequences of the viruses were determined and are presented. Further, we found hints for putative components of a methyl transferase machinery using in silico approaches. This putative protein domain is encoded by segment 2. However, this result only establishes the basis for subsequent studies in which the function must be confirmed experimentally in vitro. Thus, this is the first study where a function is predicted to all three genomic segments within the group of the alternaviruses. These findings provide further insights into the virome of ARD-associated fungi and are therefore another brick in the wall of understanding the complexity of the disease.
... The viral particle extraction, observation, and SDS-PAGE were conducted as previously described (5,55,56). Protein bands were individually excised and subjected to peptide mass analysis using mass spectrometry as described previously (57). Sequence analyses including search of homologs, ORFs, and conserved domains were performed using databases and tools in the National Center for Biotechnology Information (NCBI). ...
Mycovirus-infected fungi can suffer from poor growth, attenuated pigmentation, and virulence. However, the molecular mechanisms of how mycoviruses confer these symptoms remain poorly understood. Here, we report a mycovirus Stemphylium lycopersici alternavirus 1 (SlAV1) isolated from a necrotrophic plant pathogen Stemphylium lycopersici that causes altered colony pigmentation and hypovirulence by specifically interfering host biosynthesis of Altersolanol A, a polyketide phytotoxin. SlAV1 significantly down-regulates a fungal polyketide synthase ( PKS1 ), the core enzyme of Altersolanol A biosynthesis. PKS1 deletion mutants do not accumulate Altersolanol A and lose pathogenicity to tomato and lettuce. Transgenic expression of SlAV1 open-reading frame 3 (ORF3) in S. lycopersici inhibits fungal PKS1 expression and Altersolanol A accumulation, leading to symptoms like SlAV1-infected fungal strains. Multiple plant species sprayed with mycelial suspension of S. lycopersici or S. vesicarium strains integrating and expressing ORF3 display enhanced resistance against virulent strains, converting the pathogenic fungi into biocontrol agents. Hence, our study not only proves inhibiting a key enzyme of host phytotoxin biosynthesis as a molecular mechanism underlying SlAV1-mediated hypovirulence of Stemphylium spp., but also demonstrates the potential of mycovirus-gene integrated fungi as a potential biocontrol agent to protect plants from fungal diseases.
A novel double-strand RNA (dsRNA) mycovirus, named “Colletotrichum fioriniae alternavirus1” (CfAV1), was isolated from the strain CX7 of Colletotrichum fioriniae, the causal agent of walnut anthracnose. The complete genome of CfAV1 is composed of three dsRNA segments: dsRNA1 (3528 bp), dsRNA2 (2485 bp), and dsRNA3 (2481 bp). The RNA-dependent RNA polymerase (RdRp) is encoded by dsRNA1, while both dsRNA2 and dsRNA3 encode hypothetical proteins. Based on multiple sequence alignments and phylogenetic analysis, CfAV1 is identified as a new member of the family Alternaviridae. This is the first report of an alternavirus that infects the phytopathogenic fungus C. fioriniae.
Three dsRNA segments were detected in Fusarium pseudograminearum strain CF14029, a pathogen causing Fusarium crown rot in China. Characterization and sequence analysis confirmed that these dsRNA sequences originated from the same virus. The viral genome consists of three dsRNA segments: dsRNA1 (3,560 nt in length), encoding an RNA-dependent RNA polymerase (RdRp), dsRNA2 (2,544 nt in length), encoding a hypothetical protein, and dsRNA3 (2,478 nt in length), encoding a putative coat protein (CP). Phylogenetic analysis based on the RdRp and CP amino acid sequences revealed a high degree of similarity of this virus to members of the genus Alternavirus, family Alternaviridae, isolated from other Fusarium fungi. As a novel member of the genus Alternavirus, this virus was provisionally named "Fusarium pseudograminearum alternavirus 1" (FpgAV1). Like other alternaviruses found in Fusarium species, the positive-sense strand of each genomic dsRNA of FpgAV1 possesses a poly(A) tail and a distinctive 5'-terminal octamer sequence (5’-GCT GTG TG-3’). This is the first report of the genomic sequence of an alternavirus identified in F. pseudograminearum.
In this study, fungal spores were collected from the air, and mycelium was grown in Petri dishes containing potato dextrose agar medium in the lab. Based on the morphological and molecular characteristics, the fungus was identified as Fusarium nanum. A new double-stranded RNA (dsRNA) mycovirus was isolated from the mycelia of F. nanum and tentatively named Fusarium nanum alternavirus 1 (FnAV1). The complete genome sequence of FnAV1 was obtained by Illumina sequencing, followed by Sanger sequencing and the rapid amplification of cDNA ends (RACE) of the 5′ and 3′ ends. FnAV1 is composed of three dsRNA segments: dsRNA1 (3,546 nt) encodes RdRp, dsRNA2 (2,511 nt) encodes a hypothetical protein, and dsRNA3 encodes the coat protein (2,484 nt). The multiple alignments, along with phylogenetic analysis based on the RdRp (dsRNA1) and coat protein (dsRNA3) amino acid sequences, suggest that FnAV1 clusters with other members of the newly proposed family Alternaviridae, has high identities with Fusarium incarnatum alternavirus 1 (FiAV1), and might be a new strain isolated from a different fungal host, F. nanum. This is the first report of a mycovirus from the mycelial culture of F. nanum, which was obtained from spores collected at a 12-m altitude via air sampling. This suggests that the airborne spores could carry the mycovirus for long-distance movement and dissemination to new locations.
[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
European ash dieback caused by the alien, invasive ascomycete species Hymenoscyphus fraxineus currently represents, along with its side effects, the greatest threat to common ash (Fraxinus excelsior) trees in Europe. The disease is widely distributed in Germany and present in all regions where common ash is growing. In order to study European ash dieback in Germany and to develop management strategies to conserve common ash as a forest tree species, the concerted, interdisciplinary research project FraxForFuture was initiated. FraxForFuture consist of an association of five sub-networks: FraxConnect, FraxMon, FraxGen, FraxPath, and FraxSilva. In total, 27 individual projects conduct research on the epidemiology of the disease, the pathogen and the preservation of ash, including various control, breeding, and silvicultural strategies. The main goals of the FraxForFuture network and the sub-networks as well as essential information about the network of common research plots are provided.
Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35–193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1–4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36–50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35–193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.
A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family “Alternaviridae”. Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family “Alternaviridae” members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1—and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family “Alternaviridae”. Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family “Alternaviridae”, and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.
Biological control (BC) is defined as the use of living natural enemies, antagonists, or competitors (biological control agents) to control other living organisms. In the second half of the last century, the general interest in BC has increased considerably because greater environmental awareness in society and the implementation of integrated pest management (IPM) strategies have pushed towards the development of environmentally friendly control approaches. However, BC is still only rarely used for pathogens (fungi, bacteria, viruses, nematodes, and phytoplasmas) of forest trees. Here, we present and discuss the biological specificities of both the hosts and the fungal pathogens which may account for this situation. To increase the likelihood of BC success, we suggest a holistic approach involving the use of top-down regulators, competitors and amensalists, all exerting pressure on the pathogen, as well as bottom-up forces helping the host (e.g., endophytes, mycorrhiza). Moreover, BC to mitigate emerging forest diseases should be fully integrated into other sustainable management strategies. Finally, we propose guidelines for developing an efficient BC of emerging fungal pathogens of forest trees.
The distribution and diversity of RNA viruses in fungi is incompletely understood due to the often cryptic nature of mycoviral infections and the focused study of primarily pathogenic and/or economically important fungi. As most viruses that are known to infect fungi possess either single-stranded or double-stranded RNA genomes, transcriptomic data provides the opportunity to query for viruses in diverse fungal samples without any a priori knowledge of virus infection. Here we describe a systematic survey of all transcriptomic datasets from fungi belonging to the subphylum Pezizomycotina. Using a simple but effective computational pipeline that uses reads discarded during normal RNA-seq analyses, followed by identification of a viral RNA-dependent RNA polymerase (RdRP) motif in de novo assembled contigs, 59 viruses from 44 different fungi were identified. Among the viruses identified, 88% were determined to be new species and 68% are, to our knowledge, the first virus described from the fungal species. Comprehensive analyses of both nucleotide and inferred protein sequences characterize the phylogenetic relationships between these viruses and the known set of mycoviral sequences and support the classification of up to four new families and two new genera. Thus the results provide a deeper understanding of the scope of mycoviral diversity while also increasing the distribution of fungal hosts. Further, this study demonstrates the suitability of analyzing RNA-seq data to facilitate rapid discovery of new viruses.
Fusarium is an important genus of plant pathogenic fungi, and is widely distributed in soil and associated with plants worldwide. The diversity of mycoviruses in Fusarium is increasing continuously due to the development and extensive use of state-of-the-art RNA deep sequencing techniques. To date, fully-sequenced mycoviruses have been reported in 13 Fusarium species: Fusarium asiaticum, F. boothii, F. circinatum, F. coeruleum, F. globosum, F. graminearum, F. incarnatum, F. langsethiae, F. oxysporum, F. poae, F. pseudograminearum, F. solani, and F. virguliforme. Most Fusarium mycoviruses establish latent infections, but some mycoviruses such as Fusarium graminearum virus 1 (FgV1), Fusarium graminearum virus-ch9 (FgV-ch9), Fusarium graminearum hypovirus 2 (FgHV2), and Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1) cause hypovirulence. Rapid advances in various omics technologies used to elucidate genes or biological processes can facilitate an improved understanding of mycovirus-host interactions. The review aims to illuminate the recent advances in studies of mycoviruses in Fusarium, including those related to diversity, molecular mechanisms of virus-host interaction. We also discuss the induction and suppression of RNA silencing including the role of RNAi components as an antiviral defense response.
A novel double-stranded RNA (dsRNA) mycovirus, named “Fusarium incarnatum alternavirus 1” (FiAV1), was found in Fusarium incarnatum strain LY003-07, the causal agent of peony root rot. The complete genome of FiAV1 is composed of three dsRNA segments, dsRNA1 (3548 nt), dsRNA2 (2514 nt), and dsRNA3 (2498 nt), with one open reading frame on each of their positive-sense strands. As found in other viruses of the proposed family Alternaviridae, the positive-sense strand of each genomic dsRNA of FiAV1 has a poly(A) tail and the 5’-terminal nonamer sequence 5’-GGCTGTGTG-3’. Based on a multiple sequence alignment and phylogenetic analysis based on the RdRps amino acid sequence, FiAV1 is suggested to be a new strain of a potential new species, for which the name “Fusarium alternavirus 1” is proposed, that includes Fusarium poae alternavirus 1 (FpAV1) and Fusarium graminearum alternavirus 1 (FgAV1/AH11) of the proposed family “Alternaviridae”. This is the first report of a mycovirus of the proposed family “Alternaviridae” that infects F. incarnatum.
The molecular evolutionary genetics analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
Trunk and branch cankers are among the most important diseases compromising avocado production worldwide. A novel species, Neocosmospora perseae sp. nov. is described isolated from trunk lesions on Persea americana in the main avocado producing area of Sicily, Italy. The new species is characterised using a polyphasic approach including morphological characters and a multilocus molecular phylogenetic analysis based on partial sequences of the translation elongation factor-1α, the internal transcribed spacer regions plus the large subunit of the rDNA cistron, and the RNA polymerase II second largest subunit. Pathogenicity tests and the fulfilment of Koch's postulates confirm N. perseae as a novel canker pathogen of Persea americana.
We describe here a double-stranded RNA mycovirus, termed Fusarium graminearum alternavirus 1 (FgAV1/AH11), from the isolate AH11 of the phytopathogenic fungus F. graminearum . Phylogenetic analysis showed that FgAV1/AH11 belongs to a newly proposed family, Alternaviridae . This is the first report of a mycovirus in the family Alternaviridae that infects F. graminearum .
Fungi, similar to all species, are susceptible to viral infection. Aspergillus is arguably the most well studied fungal genus because of its medical, ecological and economical significance. Mycoviruses were initially detected in Aspergillus species almost 50 years ago and the field continues to be active today with ground-breaking discoveries. The aim of the present review is to cover the scientific progress in all aspects of mycovirology as exemplified by Aspergillus-focused research. Initially an overview of the population studies illustrating the presence of mycoviruses in numerous important Aspergillus species, such as A. niger, A. flavus, and A. fumigatus with be presented. Moreover the intricacies of mycovirus transmission, both inter- and intra-species, will be discussed together with the methodologies used to investigate viral dispersion in a laboratory setting. Subsequently, the genomic features of all molecularly characterized mycoviruses to date will be analyzed in depth. These include members of established viral families, such as Partitiviridae, Chrysoviridae and Totiviridae, but also more recent, novel discoveries that led to the proposal of new viral families, such as Polymycoviridae, Alternaviridae and, in the context of the present review, Exartaviridae. Finally, the major issue of phenotypic effects of mycoviral infection on the host is addressed, including aflatoxin production in A. flavus, together with growth and virulence in A. fumigatus. Although the molecular mechanisms behind these phenomena are yet to be elucidated, recent studies suggest that by implication, RNA silencing may be involved.
Many bands were detected on an electrophoretic profile of double-stranded (ds) RNA preparation from a single strain of Fusarium poae isolated from wheat. When the purified dsRNA sample was deep-sequenced by a next-generation sequencer, sixteen virus-like assembled contigs with predicted amino acid sequences showing homologies to respective viral RNA-dependent RNA polymerases (RdRps) were found by BLAST analysis. Fourteen out of sixteen sequences showed homologies to RdRps of known mycoviruses, that is, four mitoviruses, two narnaviruses, two partitiviruses, an alternavirus, a fusarivirus, a hypovirus, a victorivirus, and two unclassified mycoviruses, Sclerotinia sclerotiorum dsRNA mycovirus-L and Aspergillus foetidus slow virus 2, respectively. The other two putative viral RdRp sequences showed homologies to those of members of negative-stranded RNA viruses, the Ophiovirus and the Phlebovirus respectively, which mycoviruses had been not ever assigned to. Based on genome structure and phylogenetic analysis, both viruses were thought to be members of novel respective negative-stranded RNA virus groups. The presences of all sixteen viral RdRp sequences identified by BLAST analysis were confirmed by sequencing RT-PCR products generated from the starting dsRNA material using primers designed from the de novo assembled sequences of respective putative mycoviruses. Since the single strain of F. poae was considered to be multiply infected with mycoviruses from novel taxonomical groups in addition to many common mycoviruses, the RNA virome of the strain was found to be highly diverse.
Vielerorts wurden in Europa bei Eschen, die vom Eschentriebsterben (ETS) befallen waren, Stammfußnekrosen beobachtet [21]. Mit ihnen ging eine Schadensintensivierung bei den betroffenen Eschen einher [20].
Taxonomy:
Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Order Hypocreales; Family Nectriaceae; Genus Fusarium.
Host range:
Members of the FSSC collectively have a very broad host range, and have been subdivided previously into formae speciales. Recent phylogenetic analysis has revealed that formae speciales correspond to biologically and phylogenetically distinct species.
Disease symptoms:
Typically, FSSC causes foot and/or root rot of the infected host plant, and the degree of necrosis correlates with the severity of the disease. Symptoms on above-ground portions of the plant can vary greatly depending on the specific FSSC pathogen and host plant, and the disease may manifest as wilting, stunting and chlorosis or lesions on the stem and/or leaves.
Control:
Implementation of agricultural management practices, such as crop rotation and timing of planting, can reduce the risk of crop loss caused by FSSC. If available, the use of resistant varieties is another means to control disease in the field. USEFUL WEBSITES: http://genome.jgi-psf.org/Necha2/Necha2.home.html.
Unlabelled:
Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5'-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1.
Importance:
Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus (Sclerotinia sclerotiorum megabirnavirus 1 [SsMBV1]) that was isolated from an apparently hypovirulent strain, SX466, of Sclerotinia sclerotiorum. Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLPs and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses, including members of the families Potyviridae and Hypoviridae. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer might have occurred from an ssRNA virus to a dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.
A novel mycovirus, termed Fusarium graminearum Hypovirus 2 (FgHV2/JS16), isolated from a plant pathogenic fungus, Fusarium graminearum strain JS16, was molecularly and biologically characterized. The genome of FgHV2/JS16 is 12,800 nucleotides (nts) long, excluding the poly (A) tail. This genome has only one large putative open reading frame, which encodes a polyprotein containing three normal functional domains, papain-like protease, RNA-dependent RNA polymerase, RNA helicase, and a novel domain with homologous bacterial SMC (structural maintenance of chromosomes) chromosome segregation proteins. A defective RNA segment that is 4553-nts long, excluding the poly (A) tail, was also detected in strain JS16. The polyprotein shared significant aa identities with Cryphonectria hypovirus 1 (CHV1) (16.8%) and CHV2 (16.2%). Phylogenetic analyses based on multiple alignments of the polyprotein clearly divided the members of Hypoviridae into two major groups, suggesting that FgHV2/JS16 was a novel hypovirus of a newly proposed genus-Alphahypovirus-composed of the members of Group 1, including CHV1, CHV2, FgHV1 and Sclerotinia sclerotiorum hypovirus 2. FgHV2/JS16 was shown to be associated with hypovirulence phenotypes according to comparisons of the biological properties shared between FgHV2/JS16-infected and FgHV2/JS16-free isogenic strains. Furthermore, we demonstrated that FgHV2/JS16 infection activated the RNA interference pathway in Fusarium graminearum by relative quantitative real time RT-PCR.
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Members of Fusarium solani species complex (FSSC) have been known as plant, animal, and human pathogens. Nevertheless, the taxonomic status of such an important group of fungi is still very confusing and many new species as well as lineages have been elucidated recently. Unfortunately, most of the new taxa came from temperate and subtropical regions. Therefore, the objectives of the present study were to identify strains of FSSC recovered from different sources in Malaysia. In the present study, 55 strains belonging to the FSSC were examined and phylogenetically analyzed on the basis of internal transcribed spacer (ITS) regions and partial translation elongation factor-1 (TEF-1α) sequences. Based on morphological features, a total of 55 strains were selected for molecular studies. Based on morphological features, the strains were classified into four described Fusarium species, namely Fusarium keratoplasticum, Fusarium falciforme, FSSC 5, and Fusarium cf. ensiforme, and one unknown phylogenetic species was introduced. Although the data obtained from morphological and molecular studies sufficiently supported each other, the phylogenetic trees based on ITS and TEF-1α dataset clearly distinguished closely related species and distinctly separated all morphological taxa. All members of FSSC in this research were reported for the first time for Malaysian mycoflora.
The association of Fusarium graminearum isolate China-9 with the dsRNA mycovirus FgV-ch9 was evaluated for hypovirulence-related traits. Single conidia-originating cultures of China-9 isolate can be associated either with high, medium or low amounts of the viral dsRNAs. At high and medium dsRNA levels, China-9 isolates exhibit reduced mycelia growth rate and conidiation capacity, abnormal colony morphology, disorganized cytoplasm, as well as reduced virulence for wheat and maize plants. At low dsRNA levels the fungus shows no symptoms, however, the RNA segments can be detected by RT-PCR. Transfection of the virulent F. graminearum PH-1 isolate with purified Virus-like Particles (VLPs) of FgV-ch9 reduced its conidiation capacity, perithecia formation, and pathogenicity for wheat and maize several folds. These results further demonstrate that FgV-ch9 is associated with hypovirulence of F. graminearum.
We have optimized a simple and rapid preparation procedure for mushroom DNA extraction from colonies on media or from fruiting bodies for PCR amplification. The protocol combines microwaving twice for 1 min, cooling for 10 min, and centrifuging for 5 min. By using this procedure, more than 100 samples of mushroom DNA can be prepared within 1 h. The DNA obtained can be used for (1) identifying mushroom species by PCR and subsequent sequencing, (2) amplifying low copy number genes (at least 2,000 bp), and (3) screening genetic transformants. This technique will contribute to the mycology of mushroom species.
Auxotrophic, morphological and drug-resistant mutants of Cochliobolus heterostrophus (= Bipolaris maydis = Helminthosporium maydis = Drechslera maydis) were isolated after mutagenesis with ethyl methanesulphonate. Exposure of the fungus to other mutagens (N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, and UV light) resulted in recovery of morphological but not auxotrophic mutants. Antibiotic enrichment or filtration enrichment in medium containing high levels of sorbose increased the proportion of auxotrophs in the surviving population. All sexually fertile mutants were found to have single gene lesions; those with the same phenotype were allelic in some cases but not in others. Defined minimal and complete media, designed to facilitate genetic and molecular biological manipulations, supported maximal growth rate at 30 °C and maximal conidiation of the fungus at 23 °C with a 16 h long-wave UV light photoperiod. Under all in vitro growth conditions tested, near-isogenic lines of two known races of the fungus were essentially identical. Sexual fertility was improved by a programme of breeding and selection, but not by physical or chemical changes in the medium used for crossing. The fungus can be stored conveniently on silica gel.
Virus infection of Aspergillus foetidus was documented over 40 years ago and was one of the first mycovirus infections described in a filamentous fungus. The virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. Here, we report the complete nucleotide sequence of the AfV-F genome isolated from virions purified from the prototype isolate of the fungus. The AfV-F double-stranded (ds) RNA genome is tetra-segmented, and the plus strands of each of the four segments, but not the minus strands, are polyadenylated. The organisation and sequences of the four AfV-F dsRNAs are similar to those described for Alternaria alternata virus 1, which we propose is a member of an emerging mycovirus genus ("Alternavirus") and family ("Alternaviridae"), which also includes AfV-F.
Members of the species-rich Fusarium solani species complex (FSSC) are responsible for approximately two-thirds all fusarioses of humans and other animals. In addition, many economically important phytopathogenic species are nested within this complex. Due to their increasing clinical relevance and because most of the human pathogenic and plant pathogenic FSSC lack Latin binomials, we have extended the multilocus haplotype nomenclatural system introduced in a previous study (D. C. Chang, G. B. Grant, K. O'Donnell, K. A. Wannemuehler, J. Noble-Wang, C. Y. Rao, L. M. Jacobson, C. S. Crowell, R. S. Sneed, F. M. T. Lewis, J. K. Schaffzin, M. A. Kainer, C. A. Genese, E. C. Alfonso, D. B. Jones, A. Srinivasan, S. K. Fridkin, and B. J. Park, JAMA 296:953-963, 2006) to all 34 species within the medically important FSSC clade 3 to facilitate global epidemiological studies. The typing scheme is based on polymorphisms in portions of the following three genes: the internal transcribed spacer region and domains D1 plus D2 of the nuclear large-subunit rRNA, the translation elongation factor 1 alpha gene (EF-1alpha), and the second largest subunit of RNA polymerase II gene (RPB2). Of the 251 isolates subjected to multilocus DNA sequence typing, 191 sequence types were differentiated, and these were distributed among three strongly supported clades designated 1, 2, and 3. All of the mycosis-associated isolates were restricted to FSSC clade 3, as previously reported (N. Zhang, K. O'Donnell, D. A. Sutton, F. A Nalim, R. C. Summerbell, A. A. Padhye, and D. M. Geiser, J. Clin. Microbiol. 44:2186-2190, 2006), and these represent at least 20 phylogenetically distinct species. Analyses of the combined DNA sequence data by use of two separate phylogenetic methods yielded the most robust hypothesis of evolutionary relationships and genetic diversity within the FSSC to date. The in vitro activities of 10 antifungals tested against 19 isolates representing 18 species that span the breadth of the FSSC phylogeny show that members of this complex are broadly resistant to these drugs.
Four double-stranded RNAs (dsRNAs), referred to as dsRNA 1 (3617 bp), dsRNA 2 (2794 bp), dsRNA 3 (2576 bp) and dsRNA 4 (1420 bp), were detected in the EGS 35-193 strain of Alternaria alternata at high concentration ( approximately 3 microg/g dried mycelium). This strain had an impaired growth phenotype. By exposing the strain to cycloheximide during hyphal tip isolation, we isolated strains which had normal mycelial growth and pigmentation, in which decreased levels of the dsRNAs were observed ( approximately 0.3 microg/g dried mycelium). These results indicate that this dsRNA mycovirus might be involved in modulating traits of its fungal host, A. alternata. The buoyant density of isometric virus particles (about 33 nm in diameter) containing these dsRNAs in CsCl was 1.35-1.40 g/cm(3) depending on the size of the packaged dsRNAs. The dsRNA 1 encodes a single open reading frame (3447 nt) containing the conserved motifs of viral RNA-dependent RNA polymerase (RdRp), which is related to the ORF encoded by dsRNA 1 of Aspergillus mycovirus 341. It is noteworthy that all of the coding strands of the four dsRNA genomes have 3'-poly (A) tails ranging from 33 to 50 nt in length. We named this novel dsRNA mycovirus in the EGS 35-193 strain A. alternata virus-1 (AaV-1).
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
Panama disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has indicated that F. oxysporum f. sp. cubense consists of several clonal lineages that may be genetically distant. In this study we tested whether lineages of the Panama disease pathogen have a monophyletic origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1alpha and the mitochondrial small subunit ribosomal RNA genes for F. oxysporum strains from banana, pathogenic strains from other hosts and putatively nonpathogenic isolates of F. oxysporum. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The tree inferred from the combined dataset resolved five lineages corresponding to "F. oxysporum f. sp. cubense" with a large dichotomy between two taxa represented by strains most commonly isolated from bananas with Panama disease. The results also demonstrate that the latter two taxa have significantly different chromosome numbers. F. oxysporum isolates collected as nonpathogenic or pathogenic to other hosts that have very similar or identical elongation factor 1alpha and mitochondrial small subunit genotypes as banana pathogens were shown to cause little or no disease on banana. Taken together, these results indicate Panama disease of banana is caused by fungi with independent evolutionary origins.
Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.
Members of the Fusarium solani species complex (FSSC) are increasingly implicated as the causative agents of human mycoses, particularly in the expanding immunocompromised and immunosuppressed patient populations. Best known as ubiquitous plant pathogens and saprotrophs, the FSSC comprises over 45 phylogenetically distinct species distributed among three major clades. To identify which species are associated with human infections, we generated multilocus haplotypes based on four partial gene sequences from 471 isolates. Of these, 278 were from human patients, 21 were from hospital environments, and 172 were from other sources. Phylogenetic trees inferred from an ergosterol biosynthesis gene (erg-3) were highly discordant with those inferred from the three other partial gene sequences; therefore, this partition was analyzed separately. Multilocus analysis showed that isolates from humans were restricted to but spread throughout clade 3 of the FSSC phylogeny, comprising at least 18 phylogenetically distinct species. The majority (74.5%) of the clinical isolates, however, were associated with four major lineages, designated groups 1 to 4. Groups 1 and 2 were strongly supported as phylogenetic species, whereas groups 3 and 4 were not. Although isolates from ocular infections were found in all four groups, they had a significant tendency to belong to group 3 (P < 0.001). Human clinical isolates shared identical multilocus haplotypes with isolates from plants, other animals, and from hospital environments, suggesting potential nosocomiality. The major finding of this study is that FSSC-associated mycoses of humans and other animals have origins in a broad phylogenetic spectrum, indicating widespread ability to cause infection in this diverse species complex.
Fusarium keratitis is a serious corneal infection, most commonly associated with corneal injury. Beginning in March 2006, the Centers for Disease Control and Prevention received multiple reports of Fusarium keratitis among contact lens wearers.
To define the specific activities, contact lens hygiene practices, or products associated with this outbreak.
Epidemiological investigation of Fusarium keratitis occurring in the United States. A confirmed case was defined as keratitis with illness onset after June 1, 2005, with no history of recent ocular trauma and a corneal culture growing Fusarium species. Data were obtained by patient and ophthalmologist interviews for case patients and neighborhood-matched controls by trained personnel. Available Fusarium isolates from patients' clinical and environmental specimens were genotyped by multilocus sequence typing. Environmental sampling for Fusarium was conducted at a contact lens solution manufacturing plant.
Keratitis infection with Fusarium species.
As of June 30, 2006, we identified 164 confirmed case patients in 33 states and 1 US territory. Median age was 41 years (range, 12-83 years). Corneal transplantation was required or planned in 55 (34%). One hundred fifty-four (94%) of the confirmed case patients wore soft contact lenses. Forty-five case patients and 78 controls were included in the case-control study. Case patients were significantly more likely than controls to report using a specific contact lens solution, ReNu with MoistureLoc (69% vs 15%; odds ratio, 13.3; 95% confidence interval, 3.1-119.5). The prevalence of reported use of ReNu MultiPlus solution was similar between case patients and controls (18% vs 20%; odds ratio, 0.7; 95% confidence interval, 0.2-2.8). Fusarium was not recovered from the factory, warehouse, solution filtrate, or unopened solution bottles; production of implicated lots was not clustered in time. Among 39 isolates tested, at least 10 different Fusarium species were identified, comprising 19 unique multilocus genotypes.
The findings from this investigation indicate that this outbreak of Fusarium keratitis was associated with use of ReNu with MoistureLoc contact lens solution. Contact lens users should not use ReNu with MoistureLoc.
Amino acid replacement matrices are an essential basis of protein phylogenetics. They are used to compute substitution probabilities along phylogeny branches and thus the likelihood of the data. They are also essential in protein alignment. A number of replacement matrices and methods to estimate these matrices from protein alignments have been proposed since the seminal work of Dayhoff et al. (1972). An important advance was achieved by Whelan and Goldman (2001) and their WAG matrix, thanks to an efficient maximum likelihood estimation approach that accounts for the phylogenies of sequences within each training alignment. We further refine this method by incorporating the variability of evolutionary rates across sites in the matrix estimation and using a much larger and diverse database than BRKALN, which was used to estimate WAG. To estimate our new matrix (called LG after the authors), we use an adaptation of the XRATE software and 3,912 alignments from Pfam, comprising approximately 50,000 sequences and approximately 6.5 million residues overall. To evaluate the LG performance, we use an independent sample consisting of 59 alignments from TreeBase and randomly divide Pfam alignments into 3,412 training and 500 test alignments. The comparison with WAG and JTT shows a clear likelihood improvement. With TreeBase, we find that 1) the average Akaike information criterion gain per site is 0.25 and 0.42, when compared with WAG and JTT, respectively; 2) LG is significantly better than WAG for 38 alignments (among 59), and significantly worse with 2 alignments only; and 3) tree topologies inferred with LG, WAG, and JTT frequently differ, indicating that using LG impacts not only the likelihood value but also the output tree. Results with the test alignments from Pfam are analogous. LG and a PHYML implementation can be downloaded from http://atgc.lirmm.fr/LG.
While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly.
European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.
A simple method is described for designing primer sets that can amplify specific protein-encoding sequences in a wide variety of filamentous ascomycetes. Using this technique, we successfully designed primers that amplified the intergenic spacer region of the nuclear ribosomal DNA repeat, portions of the translation elongation factor 1 alpha, calmodulin, and chitin synthase 1 genes, and two other genes encoding actin and ras protein. All amplicons were sequenced and determined to amplify the target gene. Regions were successfully amplified in Sclerotinia sclerotiorum and other sclerotiniaceous species, Neurospora crassa, Trichophyton rubrum, Aspergillus nidulans, Podospora anserina, Fusarium solani, and Ophiostoma novo-ulmi. These regions are a potentially rich source of characters for population and speciation studies in filamentous ascomycetes. Each primer set amplified a DNA product of predicted size from N. crassa.
Phylogenetic relationships and biogeography of the phytopathogenic Nectria haematococca-Fusarium solani species complex, section Martiella of Fusarium, were inferred from sequence data from the nuclear large subunit 28S rDNA, the nuclear ribosomal internal transcribed spacer (ITS) region and translation elongation factor (EF-la) gene introns and exons. Although a partition homogeneity test detected significant incongruence between the 28S rDNA and ITS datasets, results of the nonparametric Wilcoxon signed-ranks Templeton test indicated that the data could be combined. Maximum parsimony analysis of the combined dataset identified 26 phylogenetically distinct ingroup species, of which 15 are heterothallic, 7 are homothallic and 4 are putatively mitosporic. Included among these were 9 formae speciales described for F. solani which were all resolved as phylogenetically distinct species by the molecular phylogeny. Of these, F. solani f. sp. cucurbitae races 1 and 2 appear to represent reproductively isolated biological species with independent evolutionary origins. Results of the molecular phylogeny also provide strong support for clades within New Zealand, South America, Africa and India-Sri Lanka. The complex historical biogeography of this complex appears to reflect vicariant events associated with the fragmentation of Gondwanaland, possible gondwanic range expansions together with relatively recent distributional changes in these fungi associated with movement of economically important plants.
In the present work, we report a novel mycovirus that infects Alternaria alternata. The mycovirus has isometric particles of approximately 30nm and the genome consists of two molecules of dsRNA, dsRNA1 with 1833bp, encoding a putative RNA-dependent RNA polymerase (RdRp) and dsRNA2, with 1680bp in length, encoding the putative capsid protein (CP). RdRp analysis revealed low amino acid identity with RdRps with species in the genus Gammapartitivirus, and the alignment of the RdRp revealed all the six conserved motifs present in members of Partitiviridae. The putative coat protein (CP) analysis revealed similarity with the putative CP of Botryosphaeria dothidea partitivirus 1 (BdPV1), a divergent partitivirus. We propose that Alternaria alternata partitivirus 1 (AtPV1) is a novel species and comprises a distinct lineage related to genus Gammapartitivirus in the family Partitiviridae, apparently on the threshold of radiation of a new genus, together with BdPV1. Vertical transmission tests showed that AtPV1 was transmitted to 100% conidial progeny and standard curing was unable to eliminate it from the host, characterizing it as a persistent virus. The absence of a virus-free isogenic lineage prevented us from accessing the details of the interaction between AtPV1 and A. alternata. Therefore, it remains unclear whether the morphological plasticity observed or the inability of the A. alternata isolate AVi1 to cause disease in plants is associated with AtPV1 infection.
The formation of collar rots in association with ash dieback was studied under different site conditions. The fungal community associated with lesions, necroses and stem collar rots, especially the occurrence of Hymenoscyphus fraxineus at these symptomatic plant tissues, was investigated. Filamentous fungi and Phytophthora spp. were isolated from affected tissues of stem collar rots of various developmental stages. Tissue samples of collar rots were collected from 32 ash trees in seven different forest plots located in Northwest Germany. Obtained isolates were assigned to morphotypes and identified based on mycelial morphology and by molecular methods. Primary agents causing collar rots were identified and the influence of site conditions was derived. The studied stem collar rots were assigned to five symptomatic categories: (0) without collar rots or lesions, (1) emerging collar rots, (2) larger collar rots without visible wood decay, (3) advanced collar rots with visible wood decay and (4) collar rots, necroses or lesions associated with dark sap oozing. In most of the studied collar rots that were collected in Schleswig- Holstein and Lower Saxony, H. fraxineus was isolated and assumed to be the primary agent. From samples of category 0 neither H. fraxineus nor other fungi or Phytophthora species were isolated. From collar rots of the other symptomatic categories, varying quantities of endophytic, saprotrophic and pathogenic species had been isolated. Overall, the number of isolated species was higher in advanced stages of collar rot. Most common species were H. fraxineus and Neonectria punicea, followed by Diaporthe eres, Botryosphaeria stevensii, Gibberella sp., Fusarium solani and Cadophora sp. However, collar rots in early stages were only associated with H. fraxineus and N. punicea. Armillaria or Phytophthora species were only isolated from advanced collar rots or occurred under special side conditions.
A novel double-stranded RNA (dsRNA) mycovirus, designated Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1), was isolated from a strain of the phytopathogenic fungus F. oxysporum f. sp. dianthi. The FodV1 genome had four dsRNA segments, designated, from the largest to the smallest one, dsRNA 1, 2 3, and 4. Each one of these segments contained a single open reading frame (ORF). dsRNA 1 (3555 bp) and dsRNA 3 (2794 bp) encoded a putative RNA-dependent RNA polymerase (RdRp) and a putative coat protein (CP), respectively. dsRNA 2 (2809 bp) and dsRNA 4 (2646 bp) contained ORFs encoding hypothetical proteins (named P2 and P4, respectively) with unknown functions. Analysis of its genomic structure, homology searches of the deduced amino acid sequences, and phylogenetic analysis all indicated that FodV1 is a new member of the family Chrysoviridae. This is the first report of the complete genomic characterization of a mycovirus identified in the plant pathogen Fusarium oxysporum.
We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a ‘universal’ primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the ‘universal’ ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.
The phylogenetic distribution of Gliocladium within the Hypocreales was investigated by parsimony analysis of partial sequences from the nuclear large subunit ribosomal DNA (28S rDNA). Two principal monophyletic groups were resolved that included species with anamorphs classified in Gliocladium. The first clade includes elements of the genera Hypocrea (H. gelatinosa, H. lutea) plus Trichoderma, Hypocrea pallida, Hypomyces and Sphaerostilbella, which are each shown to be monophyletic but whose sister group relationships are unresolved with the present data set. Gliocladium anamorphs in this clade include Gliocladium penicillioides, the type species of Gliocladium and anamorph of Sphaerostilbella aureonitens, and Trichoderma virens (= G. virens) which is a member of the clade containing Hypocrea and Trichoderma. A second clade, consisting of species with pallid perithecia, is grouped around Nectria ochroleuca, whose anamorph is Gliocladium roseum. Other species in this clade having Gliocladium-like anamorphs are Nectriopsis sporangiicola and Roumegueriella rufula. The species of Nectria represented in this study are polyphyletic and resolved as four separate groups: (1) N. cinnabarina the type species, (2) three species with Fusarium anamorphs including N. albosuccinea, N. haematococca and Gibberella fujikoroi, (3) N. purtonii a species whose anamorph is classified in Fusarium sect. Eupionnotes, and (4) N. ochroleuca, which is representative of species with pallid perithecia. The results indicate that Gliocladium is polyphyletic and that G. penicillioides, G. roseum, and Trichoderma virens (= G. virens), are generically distinct.
A large collection of strains belonging to the Fusarium solani species complex (FSSC) was isolated from soil and perithecia in primary forests in Sri Lanka (from fallen tree bark) and tropical Australia (Queensland, from fallen tree fruits and nuts). Portions of the translation elongation factor 1-alpha (tef1) gene, the nuclear large subunit (NLSU) and internal transcribed spacer regions (ITS) of the nuclear ribosomal RNA genes were sequenced in 52 isolates from soil and perithecia. The FSSC was divided previously into three clades with some biogeographic structure, termed Clades 1, 2 and 3. All Sri Lankan and Australian soil isolates were found to be members of Clade 3, most grouping with the cosmopolitan soil-associated species F. falciforme. All but two Sri Lankan perithecial isolates were associated with a set of five divergent phylogenetic lineages that were associated with Clade 2. Australian perithecial isolates resided in a subclade of Clade 3 where most of the previously defined mating populations of the FSSC reside. Isolates from perithecia and those cultured from soil were always members of different species lineages, even when derived from proximal locations. The previous biogeographic assignment of Clade 2 to South America is now expanded to the worldwide tropics. Sri Lanka appears to be an important center of diversity for the FSSC. Nectria haematococca is epitypified with a collection from the type locality in Sri Lanka; its anamorph is described as a new species, Fusarium haematococcum. Neocosmospora E.F. Smith is adopted as the correct genus for Nectria haematococca. These new species are described: F. kurunegalense/Neo. kurunegalensis, F. rectiphorus/Neo. rectiphora/, F. mahasenii/Neo. mahasenii/, F. kelerajum/Neo. keleraja.
Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.
RNA silencing can function as a virus defense mechanism in a diverse range of eukaryotes, and many viruses are capable of
suppressing the silencing machinery targeting them. However, the extent to which this occurs between fungal RNA silencing
and mycoviruses is unclear. Here, three Aspergillus dsRNA mycoviruses were partially characterized, and their relationship to RNA silencing was investigated. Aspergillus virus 1816 is related to Agaricus bisporus white button mushroom virus 1 and suppresses RNA silencing through a mechanism that alters the level of small interfering
RNA. Aspergillus virus 178 is related to RNA virus L1 of Gremmeniella abietina and does not appear to affect RNA silencing. The third virus investigated, Aspergillus virus 341, is distantly related to Sphaeropsis sapinea RNA virus 2. Detection of mycovirus-derived siRNA from this mycovirus demonstrates that it is targeted for degradation by
the Aspergillus RNA silencing machinery. Thus, our results indicate that Aspergillus mycoviruses are both targets and suppressors of RNA silencing. In addition, they suggest that the morphological and physiological
changes associated with some mycoviruses could be a result of their antagonistic relationship with RNA silencing.