Hideyuki Nagao

Random amplified polymorphic DNA analysis of japanese isolates of Verticillium dahliae and V. albo-atrum

Japanese isolates of Verticillium dahliae and V. albo-atrum have been differentiated into four and two pathogenicity groups, respectively. Japanese isolates, 29 isolates of V. dahliae and 15 isolates of V albo-atrum, representing different pathogenicity groups were analyzed by means of the random amplified polymorphic DNA (RAPD) method using polymerase chain reaction (PCR). V. dahliae could be differentiated into three subclusters and V. albo-atrum into two subclusters. In V. dahliae, the first subcluster (RAPD type I) included isolates in group A (eggplant pathotype), group C (sweet pepper pathotype), and another group with pathogenicity not determined. The second subcluster (RAPD type II) included group B isolates (tomato pathotypes). The third subcluster (RAPD type III) contained four diploid isolates (group D, brassica pathotype) and one haploid isolate (84111). In V. albo-atrum, the isolates were divided into two subclusters. The first subcluster (RAPD type IV) included isolates in only the alfalfa pathotype. The second subcluster (RAPD V) included one alfalfa pathotype isolate (Vaa-s02), three potato pathotype isolates, and three undetermined pathotype isolates from alfalfa. Similarities and differences in banding patterns obtained by RAPD could be a useful molecular tool in identification and phylogenetic studies of the pathogenicity groups.

Genetic, morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.

Supercritical Carbon Dioxide as Non-Thermal Alternative Technology for Safe Handling of Clinical Wastes

The expansions of communities and cities over the last two decades have led to the increase of the number of health care facilities, and thus, clinical wastes are generated in significant amounts. Clinical wastes are a potential source for many pathogens such as viruses, parasites, fungi and bacteria. Therefore, clinical wastes should be treated before disposal into the environment. The incineration is the most common technology applied for the treatment process. However, the negative effects of incineration on humans and the environment have led scientists to define alternative technologies for the safe disposal of clinical waste. Numerous treatment technologies have been investigated as an alternative for incineration, such as autoclave and microwave. These technologies generally depend on temperature while the recent direction is to use a non-thermal sterilization processes. SC-CO2 is one of the non-thermal sterilization technologies, which depends on pressure and low temperature. Currently, SC-CO2 has been extensively used for the inactivation of microorganisms in food and pharmaceutical industries. However, the application of SC-CO2 in treating clinical wastes has been on a rise. Studies conducted on the inactivation of fungi in food, normal saline and growth media indicate that SC-CO2 has the ability to inactivate these organisms. In clinical wastes, SC-CO2 has been found to be effective in the inactivation of pathogenic bacteria. Therefore, this review paper focuses on the potential of using SC-CO2 as alternative technology for inactivating fungi in clinical wastes.

Vegetative compatibility groups of Japanese isolates of Verticillium dahliae

Japanese isolates ofVerticillium dahliae were examined for vegetative compatibility relationships using nitrate-nonutilizing mutants. Four levels of vegetative compatibility were differentiated according to the degree of compatibility between the tester mutants ofnit1 and NitM. Wild-type growth with a complementation line greater than 5 mm wide was defined as “strong reaction (++)”, i.e., compatible. Ten out of 15 isolates showed compatibility and were separated into three groups, provisionally designated as VCGJ1, VCGJ2, and VCGJ3, depending upon their reactions. This method was used to estimate, genetic diversity within a local population ofV. dahliae. Another 12 isolates from Gunma Pref. were paired with tester isolates of the three vegetative compatibility groups proposed. Eight Gunma isolates were assigned to VCGJ1 or VCGJ2. Two isolates were incompatible with all testers. The remaining 2 isolates were self-incompatible. Thus, 18 out of 27 Japanese isolates ofV. dahliae were assigned to VCGs: 8 to VCGJ1, 7 to VCGJ2, and 3 to VCGJ3. VCGJ1 was compatible with both VCGJ2 and VCGJ3, but VCGJ2 and VCGJ3 showed a weak reaction with each other. Japanese isolates ofV. dahliae were thus demonstrated to form a VC group comprising three subgroups.

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-atrum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo‐atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo‐atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.

First report of Verticillium tricorpus isolated from potato tubers in Japan

In 1998, Verticillium sp. (CE98Vt1 and CE98Vt2) were isolated from discolored vascular structures of potato tubers sold at a market in Chiba Prefecture. These isolates were identified as Verticillium tricorpus on the basis of cultural and morphological characteristics and PCR diagnosis. This observed vascular discoloration of the potato tuber was demonstrated in three cultivars (Touya, Toyoshiro, and Waseshiro) among eight cultivars by inoculation to seedlings. External and internal symptoms of these isolates were not distinct in potato plants. The virulence of these isolates to potato was very low as compared with Verticillium dahliae. These two isolates were not pathogenic to Chinese cabbage, eggplant, green pepper, larkspur, parsley, snapdragon, soybean, tobacco, and tomato. This is the first report of V. tricorpus from potato in Japan.

How do Japanese isolates of Verticillium dahliae correspond with standardized VCG testers

We examined the vegetative compatibility of 56 Japanese isolates provisionally assigned to four subgroups ofV. dahliae to estimate the genetic relatedness with testers of the standardized VCGs. Subgroup J1 was assigned to VCG 2A/B as a new category of assignment. Subgroup J2, except isolate Vdt 110, was assigned to VCG 2A, and subgroup J3, except isolate Vdf 1, was assigned to VCG 2B. Isolates Vdf 1 and Vdt 110 were assigned to VCG 2A/B. Subgroup J4 was assigned to two subgroups, VCG 4B for Vde 1 and VCG 4A/B for FY 3 and HR 1. Four isolates were compatible with both VCG 2 and 4. Isolate U56 was compatible with VCG 2A/B and 4A. Isolates of VCG 2A, Vdt 9 and FF1, were compatible with either VCG 4A or 4A/B. One isolate of VCG 2B, Vdp-4, was compatible with VCG 4A. Three isolates of subgroup J2 showed weak reactions with the testers of VCG 4. These isolates may be “bridging strains”. Japanese isolates were composed of two VCGs, 2 and 4, “bridging strains” compatible with these VCGs, and some self-incopatible isolates. Testers of VCG 1 and VCG 3 did not show any reactions with the Japanese isolates.

The genus Thecotheus (Pezizales) in Australia: T. urinamans sp. nov. from urea-treated jarrah (Eucalyptus marginata) forest

The genus Thecotheus is reported in Australia for the first time. A new species, Thecotheus urinamans is described and illustrated and included in a key to all known species of the genus. Critical macro- and micromorphological comparisons are presented to distinguish the new species from several closely related species, particularly the widespread fungus Thecotheus crustaceus. Thecotheus urinamans was growing on rotting, moist, plant litter from an experimental plot treated with urea (ammonia) in the indigenous jarrah (Eucalyptus marginata) forest of Western Australia.

Synthesis of a Lectin in Both Mycelia and Fruit Bodies of the Ascomycete Mushroom Aleuria aurantia

In several organisms that form fruit bodies, the synthesis of lectins is developmentally regulated. Aleuria aurantia is an ascomycete that forms a fruit body known as orange peel mushroom. To find whether the mycelia of this organism synthesize a lectin, mycelial isolates were obtained from a wild fruit body by spore germination and regeneration from a fragment of the fruit body. The isolates were identified as A. aurantia by analysis of their DNA. The mycelial isolates synthesized a lectin with the same properties as those of fruit-body lectin in terms of subunit molecular mass, immunochemical reactivity, binding specificity for L-fucose, and N-terminal amino acid sequence. Vegetatively growing mycelia synthesized as much lectin as the fruit body, so such synthesis was not developmentally regulated, unlike some other organisms that form fruit bodies.

Assessment of vegetative compatibility of race-2 tomato wilt isolates of Verticillium dahliae in Japan.

Verticillium dahliae race-2 can invade the resistant cultivars of tomato possessing theVe gene. This new race was recently found in several regions in Japan, and 10 isolates ofV. dahliae race-2 from these regions were used in our study. Pathogenicity tests identified these isolates as the tomato pathotype (B). We examined the vegetative compatibility of 8 of these 10 Japanese isolates ofV. dahliae race-2 to estimate their genetic relatedness with the testers of Japanese vegetative compatibility group previously proposed (VCGJ) usingnit mutants. Compatiblenit1 and NitM mutants were obtained from allV. dahliae race-2 isolates. Selected representativenit1 and NitM mutants of eachV. dahliae race-2 isolates were paired with VCGJ testers. All isolates ofV. dahliae race-2 showed a strong reaction with VCGJ2, i.e., tomato pathotype. All isolates ofV. dahliae race-2 except for isolate To22 reacted weakly to VCGJ1 and J3. Japanese isolates ofV. dahliae race-2 were assigned as VCGJ2 and were hence vegetatively closely related with those ofV. dahliae race-1. The origin of Japanese isolates ofV. dahliae race-2 was discussed.