White Root Rot Fungus Rosellinia Research Articles

Draft Genome Sequence and Transcriptional Analysis of Rosellinia necatrix Infected with a Virulent Mycovirus.

Understanding the molecular mechanisms of pathogenesis is useful in developing effective control methods for fungal diseases. The white root rot fungus Rosellinia necatrix is a soilborne pathogen that causes serious economic losses in various crops, including fruit trees, worldwide. Here, using next-generation sequencing techniques, we first produced a 44-Mb draft genome sequence of R. necatrix strain W97, an isolate from Japan, in which 12,444 protein-coding genes were predicted. To survey differentially expressed genes (DEGs) associated with the pathogenesis of the fungus, the hypovirulent W97 strain infected with Rosellinia necatrix megabirnavirus 1 (RnMBV1) was used for a comprehensive transcriptome analysis. In total, 545 and 615 genes are up- and down-regulated, respectively, in R. necatrix infected with RnMBV1. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the DEGs suggested that primary and secondary metabolism would be greatly disturbed in R. necatrix infected with RnMBV1. The genes encoding transcriptional regulators, plant cell wall-degrading enzymes, and toxin production, such as cytochalasin E, were also found in the DEGs. The genetic resources provided in this study will accelerate the discovery of genes associated with pathogenesis and other biological characteristics of R. necatrix, thus contributing to disease control.

Differential Inductions of RNA Silencing among Encapsidated Double-Stranded RNA Mycoviruses in the White Root Rot Fungus Rosellinia necatrix.

RNA silencing acts as a defense mechanism against virus infection in a wide variety of organisms. Here, we investigated inductions of RNA silencing against encapsidated double-stranded RNA (dsRNA) fungal viruses (mycoviruses), including a partitivirus (RnPV1), a quadrivirus (RnQV1), a victorivirus (RnVV1), a mycoreovirus (RnMyRV3), and a megabirnavirus (RnMBV1) in the phytopathogenic fungus Rosellinia necatrix Expression profiling of RNA silencing-related genes revealed that a dicer-like gene, an Argonaute-like gene, and two RNA-dependent RNA polymerase genes were upregulated by RnMyRV3 or RnMBV1 infection but not by other virus infections or by constitutive expression of dsRNA in R. necatrix Massive analysis of viral small RNAs (vsRNAs) from the five mycoviruses showed that 19- to 22-nucleotide (nt) vsRNAs were predominant; however, their ability to form duplexes with 3' overhangs and the 5' nucleotide preferences of vsRNAs differed among the five mycoviruses. The abundances of 19- to 22-nt vsRNAs from RnPV1, RnQV1, RnVV1, RnMyRV3, and RnMBV1 were 6.8%, 1.2%, 0.3%, 13.0%, and 24.9%, respectively. Importantly, the vsRNA abundances and accumulation levels of viral RNA were not always correlated, and the origins of the vsRNAs were distinguishable among the five mycoviruses. These data corroborated diverse interactions between encapsidated dsRNA mycoviruses and RNA silencing. Moreover, a green fluorescent protein (GFP)-based sensor assay in R. necatrix revealed that RnMBV1 infection induced silencing of the target sensor gene (GFP gene and the partial RnMBV1 sequence), suggesting that vsRNAs from RnMBV1 activated the RNA-induced silencing complex. Overall, this study provides insights into RNA silencing against encapsidated dsRNA mycoviruses. Encapsidated dsRNA fungal viruses (mycoviruses) are believed to replicate inside their virions; therefore, there is a question of whether they induce RNA silencing. Here, we investigated inductions of RNA silencing against encapsidated dsRNA mycoviruses (a partitivirus, a quadrivirus, a victorivirus, a mycoreovirus, and a megabirnavirus) in Rosellinia necatrix We revealed upregulation of RNA silencing-related genes in R. necatrix infected with a mycoreovirus or a megabirnavirus but not with other viruses, which was consistent with the relatively high abundances of vsRNAs from the two mycoviruses. We also showed common and different molecular features and origins of the vsRNAs from the five mycoviruses. Furthermore, we demonstrated the activation of RNA-induced silencing complex by mycoviruses in R. necatrix Taken together, our data provide insights into an RNA silencing pathway against encapsidated dsRNA mycoviruses which is differentially induced among encapsidated dsRNA mycoviruses; that is, diverse replication strategies exist among encapsidated dsRNA mycoviruses.

A Novel Victorivirus from a Phytopathogenic Fungus, Rosellinia necatrix, Is Infectious as Particles and Targeted by RNA Silencing

A novel victorivirus, termed Rosellinia necatrix victorivirus 1 (RnVV1), was isolated from a plant pathogenic ascomycete, white root rot fungus Rosellinia necatrix, coinfected with a partitivirus. The virus was molecularly and biologically characterized using the natural and experimental hosts (chestnut blight fungus, Cryphonectria parasitica). RnVV1 was shown to have typical molecular victorivirus attributes, including a monopartite double-stranded RNA genome with two open reading frames (ORFs) encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), a UAAUG pentamer presumed to facilitate the coupled termination/reinitiation for translation of the two ORFs, a spherical particle structure ~40 nm in diameter, and moderate levels of CP and RdRp sequence identity (34 to 58%) to those of members of the genus Victorivirus within the family Totiviridae. A reproducible transfection system with purified RnVV1 virions was developed for the two distinct fungal hosts. Transfection assay with purified RnVV1 virions combined with virus elimination by hyphal tipping showed that the effects of RnVV1 on the phenotype of the natural host were negligible. Interestingly, comparison of the RNA silencing-competent (standard strain EP155) and -defective (Δdcl-2) strains of C. parasitica infected with RnVV1 showed that RNA silencing acted against the virus to repress its replication, which was restored by coinfection with hypovirus or transgenic expression of an RNA silencing suppressor, hypovirus p29. Phenotypic changes were observed in the Δdcl-2 strain but not in EP155. This is the first reported study on the host range expansion of a Totiviridae member that is targeted by RNA silencing.

Extending the Fungal Host Range of a Partitivirus and a Mycoreovirus from Rosellinia necatrix by Inoculation of Protoplasts with Virus Particles

The potential host range of mycoviruses is poorly understood because of the lack of suitable inoculation methods. Recently, successful transfection has been reported for somatically incompatible fungal isolates with purified virus particles of two mycoviruses, the partitivirus RnPV1-W8 (RnPV1) and the mycoreovirus RnMyRV3/W370 (MyRV3), from the white root rot fungus Rosellinia necatrix (class Sordariomycetes, subclass Xylariomycetidae). These studies examined and revealed the effect of the mycoviruses on growth and pathogenicity of R. necatrix. Here, we extended the experimental host range of these two mycoviruses using a transfection approach. Protoplasts of other phytopathogenic Sordariomycetous fungi-Diaporthe sp., Cryphonectria parasitica, Valsa ceratosperma (Sordariomycetidae), and Glomerella cingulata (Hypocreomycetidae)-were inoculated with RnPV1 and MyRV3 viral particles. The presence of double-stranded RNA viral genomes in regenerated mycelia of Diaporthe sp., C. parasitica, and V. ceratosperma confirmed both types of viral infections in these three novel host species. An established RnPV1 infection was confirmed in G. cingulata but MyRV3 did not infect this host. Horizontal transmission of both viruses from newly infected strains to virus-free, wild-type strains through hyphal anastomosis was readily achieved by dual culture; however, vertical transmission through conidia was rarely observed. The virulence of Diaporthe sp., C. parasitica, and V. ceratosperma strains harboring MyRV3 was reduced compared with their virus-free counterpart. In summary, our protoplast inoculation method extended the experimental host range of RnPV1-W8 and MyRV3 within the class Sordariomycetes and revealed that MyRV3 confers hypovirulence to the new hosts, as it does to R. necatrix.

Hot water treatment of Japanese pear trees is effective against white root rot caused by Rosellinia necatrix Prillieux

Hot water was dripped into the rhizosphere of Japanese pear trees (Pyrus serotina Rehd. grafted on P. betulifolia Bunge.) infested with the white root rot fungus Rosellinia necatrix Prillieux, to destroy the fungus. Isolates of R. necatrix from diseased roots of Japanese pear were vulnerable to water at temperatures above 35°C, and the fungus was eradicated from the colonized substrate when water at 35°C was provided for 3 days. The time required to eradicate R. necatrix decreased exponentially with increasing temperature. Japanese pear trees tolerated a temperature of 45°C without reduction in vigor. Field experiments demonstrated the practical use of hot water drip irrigation (HWD). HWD at 50°C completely destroyed white root rot mycelia on diseased roots, and many rootlets grew after the treatment. HWD at this temperature caused no injury to the trees. HWD of diseased orchard trees was assessed in Takamori and Iida in southern Nagano, Japan. The fungus recurred in two of four trees 28 months after treatment in Takamori and in two of ten trees 16 months after treatment in Iida. The new mycelia emerged on thick roots deep within the soil. Although there is a possibility of recurrence, HWD treatment is a practical control measure for white root rot.

Agrobacterium tumefaciens -mediated genetic transformation of the white root rot ascomycete Rosellinia necatrix

Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker gene were demonstrated by Southern analysis. This is the first report describing A. tumefaciens-mediated transformation of R. necatrix

Primary structure of cytochrome c gene from the white root rot fungus Rosellinia necatrix.

The nucleotide sequence of the cytochrome c (CytC) gene of the white root rot fungus Rosellinia necatrix was analyzed. The structure of this gene, which had three introns in the coding region, was similar to that of Aspergillus nidulans. The second intron of the R. necatrix CytC gene was not present in Neurospora crassa or Fusarium oxysporum. However, the amino acid sequence of R. necatrix was most similar to that of Neurospora crassa. Thus, it seemed that the second intron of the R. necatrix CytC gene was inserted into its present position after R. necatrix and its closest relatives diverged evolutionarily.

Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000bp to more than 10kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs.

Nucleotide sequences of double-stranded RNA segments from a hypovirulent strain of the white root rot fungus Rosellinia necatrix: possibility of the first member of the Reoviridae from fungus.

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41 x 10(6) to 2.95 x 10(6). Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5'-end (5'-ACAAUUU-3') and at the 3'-end (5'-UGCAGAC-3') were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.

Observations on the teleornorph of the white root rot fungus, Rosellinia necatrix, and a related fungus, Rosellinia aquila

The process of teleomorph development in the white root rot fungusRosellinia necatrix is described on diseased roots of Japanese pear. Stromata were also found on dead plants in nonagricultural lands such as yards and forests. The stroma ofR. aquila is also described.