Molecular Characterization of <Emphasis Type="Italic">Bean yellow mosaic virus </Emphasis>from Vegetatively Propagated Dwarf <Emphasis Type="Italic">Gentiana </Emphasis>Plants

The causative virus (isolate No. 4) of gentian (Gentiana spp.) mosaic, which had been identified previously as Clover yellow vein virus (C1YVV) on the basis of host range and serological reactions, was re-identified as Bean yellow mosaic virus (BYMV) on the basis of the nucleotide sequences of the gene for the coat protein (CP) and the 3′-noncoding region, as well as the predicted amino acid sequence of CP. Received 16 April 2002/ Accepted in revised form 19 June 2002     

Neutralizing test of hemagglutinating encephalomyelitis virus (HEV) in FS-L3 cells cultured without serum

FS-L3 cells, originating from porcine kidney, were used for propagation of Hemagglutinating encephalomyelitis virus (HEV) and development of a virus neutralizing (VN) test. Sera of pigs, rats, cows and dogs had VN activities to HEV. On the other hand, sera of mice, rabbits, goats, sheep, horses, cats, chickens, hamsters and human did not have measurable VN activities, although these sera had high HI activities. Our results support the idea that the VN is a more reliable measure of HEV infection than the conventionally used HI test.     

Further evidence of Mirafiori lettuce big-vein virus but not of Lettuce big-vein associated virus with big-vein disease in lettuce

Mirafiori lettuce big-vein virus (MLBVV) and Lettuce big-vein associated virus (LBVaV) are found in association with big-vein disease of lettuce. Discrimination between the two viruses is critical for elucidating the etiology of big-vein disease. Using specific antibodies to MLBVV and LBVaV for western blotting and exploiting differences between MLBVV and LBVaV in host reaction of cucumber and temperature dependence in lettuce, we separated the two viruses by transfering each virus from doubly infected lettuce plants to cucumber or lettuce plants. A virus-free fungal isolate was allowed to acquire the two viruses individually or together. To confirm the separation, zoospores from MLBVV-, LBVaV-, and dually infected lettuce plants were used for serial inoculations of lettuce seedlings 12 successive times. Lettuce seedlings were infected at each transfer either with MLBVV alone, LBVaV alone, or both viruses together, depending on the virus carried by the vector. Lettuce seedlings infected with MLBVV alone developed the big-vein symptoms, while those infected with LBVaV alone developed no symptoms. In field surveys, MLBVV was consistently detected in lettuce plants from big-vein-affected fields, whereas LBVaV was detected in lettuce plants not only from big-vein-affected fields but also from big-vein-free fields. LBVaV occurred widely at high rates in winter-spring lettuce-growing regions irrespective of the presence of MLBVV and, hence, of the presence of the big-vein disease.     

Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease

Research on Olpidium virulentus, the vector of the serious lettuce big-vein disease, is difficult because its resting spores persist in soil and retain virus for years and the fungus is not culturable. We developed protocols to obtain purified, viable resting spores from infected roots using an enzymatic treatment and two-step density gradient centrifugation and to generate a polyclonal antibody against these spores. The antibody was species-specific in a direct immunostaining assay and in western blots produced two specific bands (ca. 30.5, 29.0 kDa) against resting spores. The detection limit for resting spores in an indirect ELISA was 200 spores/100 μL.     

Diversity of rDNA-ITS sequences of <Emphasis Type="Italic">Polymyxa graminis</Emphasis> from wheat and barley in Japan

Polymyxa graminis is a soil-borne obligate organism that transmits bymoviruses and furoviruses to barley and wheat. We analyzed rDNA-ITS sequences of P. graminis from roots of wheat and barley in fields in Japan and obtained five kinds of sequences; two sequences were almost the same as known ribotype Ia and IIa, respectively, and three were close to ribotype Ib. When infection of P. graminis was examined using PCR, ribotype Ia was detected only in barley, but ribotypes Ib and IIa were detected in both wheat and barley. Our analysis suggested that Japanese ribotype Ib transmits furoviruses and bymoviruses.     

Seasonal influence on testicular function of male raccoons, Procyon lotor

In order to clarify the breeding capability of male raccoons in Japan, the testes of raccoons, a nuisance animal, collected in Kanagawa Prefecture and Hokkaido were histologically inspected. Furthermore, testosterone concentrations in their blood were measured. The testosterone concentrations increased in winter and the diameter of seminiferous tubules and the spermatogenetic score decreased in summer for the animals captured in Kanagawa Prefecture. For the animals captured in Hokkaido, the diameter of seminiferous tubules did not change and the decrease of the spermatogenetic score in summer was slight. As the above results show, there is a breeding season in male raccoons in Japan, and the reduction of testicular function in summer was greater in animals captured in Kanagawa Prefecture than in animals captured in Hokkaido.     

Biological, serological, and molecular variabilities of clover yellow vein virus

ABSTRACT A comparative study was made on the host reactions, serological properties, and nucleotide sequences of the coat protein (CP) gene of 10 clover yellow vein virus (C1YVV) isolates and one bean yellow mosaic virus (BYMV) isolate collected from different host plant species and locations in Japan. Two strains of C1YVV isolates, grouped on the basis of host reactions on Chenopodium amaranticolor, C. quinoa, Nicotianaclevelandii, N. benthamiana, Vicia faba, and Trifolium repens, corresponded to two serotypes determined by double-antibody sandwich- and triple-antibody sandwich-enzyme-linked immunosorbent assay using three polyclonal and nine monoclonal antibodies. These results were also confirmed by nucleotide sequence analysis of the CP gene. The CP gene of C1YVV isolates of strain 1, including the Australian isolate C1YVV-B, had 93 to 98% nucleotide identities and 97 to 99.6% amino acid identities. The CP of C1YVV isolates of strain 2, including the New Zealand isolate C1YVV-NZ, had 92 to 98% nucleotide identities and 95 to 98% amino acid identities. The nucleotide identities and the amino acid identities between the two C1YVV strains were 82 to 84%, and 90 to 94%, respectively. When compared with the CP sequences of 12 C1YVV isolates, the CP sequence of the BYMV isolate had 71 to 73% nucleotide identity and 73 to 77% amino acid identity. Amino acid sequence differences among C1YVV isolates from strains 1 and 2 were located mostly at the N-terminal regions of the CP. Our results indicated that the C1YVV isolates studied could be separated into two strains on the basis of host reactions, serology, and the nucleotide sequence of the CP gene.     

Virulence characteristics of Yersinia pseudotuberculosis isolated from breeding monkeys in Japan

Between April 2001 and 2007, 18 Yersinia pseudotuberculosis outbreaks occurred in breeding monkeys at 12 zoological gardens in Japan, and 28 monkeys of 8 species died. A total of 18 Y. pseudotuberculosis strains from the dead monkeys, comprising one strain per outbreak, were examined for serotype and the presence of the virulence genes virF, inv, ypm (ypmA, ypmB and ypmC) and irp2. Of the 18 Y. pseudotuberculosis strains, 7 (38.9%) were serotype 4b, 7 (38.9%) were serotype 1b, and there was one each of serotypes 2b, 3, 6 and 7. All the 18 strains examined harbored virF and inv. Sixteen (88.9%) strains, including the strain of serotype 7, harbored ypmA. However, no strain harbored ypmB, ypmC and irp2.

This study demonstrated that among other pathogenic factors, almost all the Y. pseudotuberculosis isolated from the outbreaks had the ypm gene encoding the superantigenic toxin, YPM. As most of the monkeys who died in those outbreaks originated from South America and other regions, where the presence of the ypm gene have not been reported, YPM might be the cause, or at least the most important factor for, the high mortality of the breeding monkeys infected by Y. pseudotuberculosis in Japan. This is also the first report of a fatal case due to Y. pseudotuberculosis serotype 7 infection in the world.     

No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types

In boreal forests ericaceous shrubs often dominate the forest floor vegetation. Nitrogen enrichment has been shown to decrease shrub abundance and in this study we explored whether it also affects the root associated fungal communities. Fine roots of Vaccinium myrtillus were collected in a Norway spruce dominated forest and of Vaccinium vitis-idaea in a Scots pine dominated forest. In both forests, nitrogen enrichment was experimentally induced by adding 12.5 and 50 kg N ha⁻¹ yr⁻¹ for 12 (spruce forest) and four (pine forest) years. Based on terminal restriction fragment length polymorphisms, subcloning and sequencing analyses, the root associated fungal communities were examined. We found 93 fungal species including Asco-, Basidio- and Zygo-mycota. In general, the Rhizoscyphus ericae aggregate was the most dominant and this was followed by Herpotrichiellaceae and Sebacina. Ordination analysis revealed that nitrogen enrichment did not change species composition of the fungal communities in neither the spruce nor the pine forest, while fungal community structures were clearly discriminated between the dominant shrub species in each forest. Similarly, no fungal species showed a significant response to nitrogen enrichment. Therefore, nitrogen enrichment appears to have no effect on root associated fungi of understorey dwarf shrubs in boreal forests, while it is clear that spruce and pine forests harbor distinctive communities of these fungi.