Yukari Uetake

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.

Ultrastructural changes during the symbiotic development of Spiranthes sinensis (Orchidaceae) protocorms associated with binucleate Rhizoctonia anastomosis group C

The process of symbiotic development of Spiranthes sinensis seeds to protocorms with differentiated shoots associated with binucleate Rhizoctonia anastomosis group C was divided into 7 stages (I–VII) based on increase of embryo and protocorm width. Each stage was observed ultrastructurally. The embryo contained large amount of lipid, small amount of starch, and protein body-like structure in all its cells. The embryo began to swell (stage I) as soon as several hyphae invaded the basal cells. Hyphae penetrated into the inner cortical parenchyma (ICP) and subepidermal parenchyma (SEP) cells and formed pelotons. The meristematic region (MR) was not colonized. Hyphal cell walls (FCW) were surrounded by an encasement layer (EL) and by the host plasmalemma. Pelotons that formed in ICP had thin walls and were digested, whereas pelotons in SEP were not digested. In stage I, mitochondria, ribosomes, proplastids, microbodies, rough endoplasmic reticulum, dictyosomes and vacuoles were observed in the host cytoplasm. Signs of hyphal digestion was already apparent in this stage. In stage II, clumps of digested pelotons consisted only of layers of FCW and EL and were surrounded by host cytoplasm and vacuoles, and/or secondary infected hyphae. Hyphal digestion and the reinfection of host cells occurred repeatedly throughout all growth stages. Protein body-like structures rapidly disappeared before stage III. At stage V and subsequent stages, it was also seen that the dispersed contents of digested hyphae were surrounded by the host plasmalemma. When ICP cells contained pelotons, amyloplasts were not observed. However, proplastids usually existed in the cells, and amyloplasts were observed in all SEP cells and other uninfected cells. Lipid bodies were slowly degraded throughout embryo and protocorm growth.

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.

Cytochemical localization of adenylate cyclase activity in the symbiotic protocorms of Spiranthes sinensis

A cytochemical method has been used to investigate the localization of adenylate cyclase [EC 4.6.1.1] activity in the protocorms of Spiranthes sinensis developing with one of its endomycorrhizal fungi, Ceratobasidium cornigerum and in seeds cultured without fungi. In both symbiotic protocorms and asymbiotic seeds, the reaction products precipitated on the exterior side of the host plasma membrane in contact with the host cell wall. The invagination sites of the plasma membrane and vesicles near the membrane showed a strong reaction. Non-electron-dense deposits were found on the host membrane enveloping the hyphae and on the fungal plasma membrane. The host membrane enveloping the hyphae stained with the two selective stains for plasma membrane of plant cells, phosphotungstic acid-chromic acid and alkaline bismuth. The detection of the adenylate cyclase activity on the host plasma membrane in contact with host cell wall suggested the presence of cAMP in orchid tissues, and the absence of activity on the host plasma membrane enveloping the hyphae suggested that cAMP played no role in the interface between host and fungus.

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.

Spatial associations between actin filaments, endoplasmic reticula, mitochondria and fungal hyphae in symbiotic cells of orchid protocorms

The relationships between endoplasmic reticula (ER), mitochondria, and actin filaments (Afs) were observed in uncolonized and colonized cells of symbiotic protocorms ofSpiranthes sinensis (Orchidaceae) germinated in the presence of the fungus,Ceratobasidium cornigerum. Mitochondria and ER were observed by transmission electron microscopy, and with the fluorescent probe DiOC6 (3) (3,3′-dihexyloxacarbocyanine) combined with confocal laser scanning microscopy (CLSM). An indirect immunofluorescence method using CLSM and an indirect, pre-embedding immunogold method at the ultrastructural level were used for observation of Afs. In uncolonized cells, cortical ER showed a polygonal pattern and ER formed a network throughout the cytoplasm. In the cortex, a smooth face of ER contacted the plasma membrane. Mitochondria were associated with ER. Afs were in close proximity to ER, mitochondria and amyloplasts. Colonized cells retained cortical ER, and a smooth face of ER was also closely associated with the perifungal membrane. ER and mitochondria were present in the cytoplasmic channels bridging between the central peloton and the peripheral cytoplasm. This distribution of ER and mitochondria during fungal colonization and senescence coincided with that of Afs. The changes in the arrays of Afs accompanying symbiotic fungal colonization and senescence occurred concomitantly with the changes in ER.

Helicobasidium mompa isolates from sweet potato in continuous monoculture fields

Abstract Isolates of the violet root rot fungus Helicobasidium mompa were collected from herbaceous and tree plants. Their host preference was studied by inoculation experiments using carrots, sweet potatoes, and apple stocks. It was found that sweet potato isolates from Kyushu produced infection cushions on carrots and sweet potatoes but not on apple stocks. Other isolates did not show host preference. Sweet potato isolates were also characterized by ready hyphal mass (sclerotium) production. They were thought to have adapted to the habitat with high disturbance by annual tillage.

An Improved Method for Isolating Violet Root Rot Fungus, Helicobasidium mompa, from Basidiocarps

Basidiocarps of the violet root rot fungus, Helicobasidium mompa, are less frequently used for isolation than are mycelial strands and sclerotia even though the basidiocarps are conspicuously produced at the trunk base of diseased plants. Basidiocarps are also more suitable for storage. This paper describes an improved method for obtaining pure cultures from basidiocarps using microcentrifuge tubes to facilitate the awkward steps of rinsing fungal materials under a dissecting microscope.