Masao Arakawa

A Reovirus Causes Hypovirulence of Rosellinia necatrix.

ABSTRACT White root rot, caused by Rosellinia necatrix, is a serious soilborne disease of fruit trees and other woody plants. R. necatrix isolate W370 contains 12 segments of double-stranded RNA (dsRNA) that is believed to represent a possible member of the family Reoviridae. W370 was weakly virulent and its hyphal-tip strains became dsRNA free and strongly virulent. The 12 segments of W370dsRNA were transmitted to hygromycin B-resistant strain RT37-1, derived from a dsRNA-free strain of W370 in all or none fashion through hyphal contact with W370. The W370dsRNA-transmitted strains were less virulent than their parent strain RT37-1 on apple seedlings, with mortality ranging between 0 to 16.7% in apple seedlings that were inoculated with the W370dsRNA-containing strains and 50 to 100% for seedlings inoculated with the dsRNA-free strains. Some W370dsRNA-containing strains killed greater than 16.7% of seedlings, but these were found to have lost the dsRNA in planta. These results indicate that W370dsRNA is a hypovirulence factor in R. necatrix. In addition, a strain lost one segment (S8) of W370dsRNA during subculture, and the S8-deficient mutant strain also exhibits hypovirulence in R. necatrix.

The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

Summary.The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

Diversity and vertical transmission of double-stranded RNA elements in root rot pathogens of trees, Helicobasidium mompa and Rosellinia necatrix.

The diversity and vertical transmission of double-stranded (ds) RNA in Helicobasidium mompa and Rosellinia necatrix was examined by electrophoresis and Northern hybridization. These two fungi share the similar niche as root rot pathogens of trees in forests and orchards, and had diverse dsRNAs. The detection frequency of dsRNA in both fungi was different; in H. mompa, 68.4% (132 out of 193 MCGs; mycelial compatibility groups) had dsRNA, whereas 20.9% (53 out of 254 MCGs) in R. necatrix. dsRNA banding patterns and Northern blot analyses revealed the presence of various dsRNA elements in both fungi. Hyphal tip isolation was mostly unsuccessful to remove dsRNA with some exceptions. Sexual reproduction functioned to remove dsRNA in both fungi since dsRNA was not detected from single sexual spore cultures. Possible explanations for the difference in the detection frequency of dsRNA are discussed in terms of the differences in their sexual reproduction and other factors.

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 0.41×106 to 2.95×106. 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 teleomorph 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.

ITS polymorphism within a single strain of Sclerotium rolfsii

Two morphologically distinct strains, 63–76 and 63H1, were isolated from a protoplast and a hyphal tip of the parentalSclerotium rolfsii strain S-63, respectively. Strains 63–76 and 63H1 showed reduced mycelial growth and lacked clamp connections on hyphae. The two strains also differed from each other and from their parent in RAPD patterns generated by several primers, suggesting that 63–76 and 63H1 were homokaryons isolated from the hetereokaryon S-63. Whereas the parent S-63 belonged to ITS-RFLP group 1, RFLP patterns of internal transcribed spacer (ITS) regions of rDNA of 63–76 and 63H1 were similar to those of ITS-RFLP groups 5 and 3, respectively. The sequence similarity of ITS regions were more than 99% between 63–76 and group 5 strains, 100% between 63H1 and the group 3 strain, and 96.3% between 63–76 and 63H1. Direct sequencing failed in the parental strain S-63. S-63 was considered to contain ITS types of groups 5 and 3.

Conidioma production of the white root rot fungus in axenic culture under near-ultraviolet light radiation

Abstract Conidiomata of the white root rot fungus were produced in axenic culture under near-ultraviolet light radiation. Pieces of sterilized Japanese pear twigs were placed on 7-day-old oatmeal agar culture in plates. The plates were further incubated for 5 days and then illuminated by near-ultraviolet light. Synnemata developed on the twigs within 5 weeks in 19 of 20 isolates tested, and conidia were observed in 12 of the 19 isolates. The synnemata and conidia produced were morphologically identical to those of Dematophora necatrix.

Inoculation of Lupinus luteus with White Root Rot Fungus, Rosellinia necatrix, to Estimate Virulence

An inoculation method using Lupinus luteus was developed for estimating virulence of isolates of the white root rot fungus, Rosellinia necatrix Prillieux. Fungal cultures grown on pieces of mulberry twigs were placed in contact with the hypocotyl of 3-week-old seedlings growing in pots of soil. Disease development was uniform and reproducible in repeated experiments. Of 24 isolates with double-stranded RNA, eight were weakly virulent. This method is useful throughout the year for estimating virulence of many isolates of the fungus and for screening for hypovirulent isolates.

Genetic relationships among violet root rot fungi as revealed by hyphal anastomosis and sequencing of the rDNA ITS regions

Genetic relationships among the violet root rot fungi were studied by hyphal anastomosis and sequencing of the ITS-5.8S rDNA regions. Twelve isolates of Helicobasidium mompa, one isolate each of H. purpreum and H. compactum, two isolates of Helicobasidium sp., eight isolates of Rhizoctonia crocorum, and three isolates of R. violacea anastomosed in most combinations, resulting in the death of fused cells and adjacent cells. Phylogenetic analyses of the ITS-5.8S rDNA regions separated 31 violet root rot fungal isolates into five lineages: H. mompa from Japan and Korea; R. crocorum from Europe and New Zealand; H. purpureum, R. crocorum, and R. violacea from Europe; Helicobasidium sp. from Japan; and H. compactum from the USA. Within each lineage there was no more than one nucleotide substitution, and similarity ranged from 81.8% to 98.2% between lineages. The discrepancy between the results from the two methods are discussed in terms of the biological species concept in the complex.

A Root Box Method to Estimate Virulence of Helicobasidium mompa Using Carrots and Its Comparison with the Conventional Method Using Apple Stocks

A root box method with carrots was developed to estimate virulence of the violet root rot fungus, Helicobasidium mompa, to facilitate short-term screening of many isolates during a year. The root box consisted of two transparent acrylic plates and a plastic bag of vermiculite in which two taproots of carrot were growing and inoculated with the fungus growing on fragments of mulberry twigs. The boxes were kept in a greenhouse at 25°C, and the surface of carrots was observed weekly up to 14 weeks. The virulence of each isolate was determined based on the number of weeks after inoculation required for the fungus to develop infection cushions on the surface of carrots. Results were compared with those from the conventional inoculation method using apple stocks. Two-year-old 456 apple stocks were planted with or without fungal inoculum in 30-cm diam. plastic pots containing commercial soil and placed outdoors in April 1999. Symptoms on plant tops were observed weekly, and the first stocks were killed 14 weeks after inoculation. At the end of trial 1 (6 months) and trial 2 (14 months), apple stocks were dug up to rate disease index (DI) based on hyphal growth and infection cushion formation on the stem base. There was variability in disease severity among replicates as well as isolate variability ; however, the results were similar in both trails. The level of virulence estimated by both methods was almost parallel for a total of 23 isolates from five plant species, except for two isolates from sweet potato that formed no obvious infection cushion on apple roots but on carrot were the most virulent.