Spray Application of Nonpathogenic Fusaria onto Rice Flowers Controls Bakanae Disease (Caused by Fusarium fujikuroi) in the Next Plant Generation.

Bakanae disease, caused by Fusarium fujikuroi, is an economically important seed-borne disease of rice. F. fujikuroi is horizontally transmitted to rice flowers and vertically transmitted to the next generation via seeds. The fungus induces typical symptoms such as abnormal tissue elongation and etiolation. Sanitation of seed farms and seed disinfection are the only effective means to control bakanae disease at present; however, the efficacy of these methods is often insufficient. Therefore, alternative and innovative control methods are necessary. We developed a novel method for applying nonpathogenic fusaria as biocontrol agents by spraying spore suspensions onto rice flowers to reduce the incidence of seed-borne bakanae. We visualized the interaction between Fusarium commune W5, a nonpathogenic fusarium, and Fusarium fujikuroi using transformants expressing two different fluorescent proteins on/in rice plants. W5 inhibited hyphal extension of F. fujikuroi on/in rice flowers and seedlings, possibly by competing with the pathogen, and survived on/in rice seeds for at least 6 months.IMPORTANCE We demonstrated that a spray treatment of rice flowers with the spores of nonpathogenic fusaria mimicked the disease cycle of the seed-borne bakanae pathogen Fusarium fujikuroi and effectively suppressed the disease. Spray treatment of nonpathogenic fusaria reduced the degree of pathogen invasion of rice flowers and vertical transmission of the pathogen to the next plant generation via seeds, thereby controlling the bakanae disease. The most promising isolate, F. commune W5, colonized seeds and seedlings via treated flowers and successfully inhibited pathogen invasion, suggesting that competition with the pathogen was the mode of action. Seed-borne diseases are often controlled by seed treatment with chemical fungicides. Establishing an alternative method is a pressing issue from the perspectives of limiting fungicide resistance and increasing food security. This work provides a potential solution to these issues using a novel application technique to treat rice flowers with biocontrol agents.

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans.

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Biolistic inoculation of soybean plants with soybean dwarf virus.

Soybean dwarf virus (SbDV), a member of the genus Luteovirus, has been transmitted only by aphid vectors. The possibility of using a biolistic procedure of transmitting SbDV to soybean plants without relying on aphid vectors was investigated. Biolistic inoculation using the Helios Gene Gun System with RNA transcribed in vitro from a full-length cDNA clone of the DS strain of SbDV (pSV-DS) resulted in 1/12 to 3/13 soybean plants infected systemically. The infected soybean plants showed the characteristic symptoms of SbDV-DS within 6 weeks after inoculation and the accumulation of SbDV-specific RNA species such as genomic and subgenomic RNAs in the upper non-inoculated leaves. The progeny virus derived from RNA transcribed in vitro from pSV-DS could be transmitted by aphid vectors, as is the case with native SbDV-DS. This is the first report of direct inoculation of soybean plants with SbDV without using aphid vectors.

Discrimination of four soybean dwarf virus strains by dot-blot hybridization with specific probes.

Soybean dwarf virus (SbDV) is divided into four strains (YS, YP, DS, and DP) on the basis of host symptoms in infected soybean plants and on aphid vector specificity. To detect and discriminate each strain of SbDV by dot-blot hybridization, probes Y, D, S, and P were prepared. Probes Y and D, covering most of the 3'-noncoding region of the viral genome containing the sequence of small subgenomic RNA, could discriminate strains in accord with the host symptoms. Probes S and P were derived from the 5'-half of open reading frame 5 encoding the N-terminal half of the readthrough domain which is closely related to the aphid vector specificity of each strain. Thus, the four SbDV strains could be discriminated by the combination of these probes. This method, based on a procedure specific to the SbDV sequence, is a good alternative for routine examination of infected plants in soybean breeding programs for evaluation of resistance to SbDV and for assessment of the distribution of each strain in epidemiological studies.

The N-Terminal Region of the Readthrough Domain Is Closely Related to Aphid Vector Specificity of Soybean dwarf virus.

ABSTRACT It has been speculated that the N-terminal half of the readthrough domain (RTD) encoded by open reading frame 5 of Soybean dwarf virus (SbDV) is related to the vector specificity. To further investigate this hypothesis, transmissibility via aphids was tested on 17 SbDV isolates and comparisons of the deduced amino acid sequences of the coat protein (CP) and other proteins encoded by the RTD were made between these isolates. Isolates were distinguished into four strains: YS, causing yellowing in soybean and transmittable by Aulacorthum solani; DS, causing dwarfing and transmittable by A. solani; YP, causing yellowing and transmittable by Acyrthosiphon pisum; and DP, causing dwarfing and transmittable by A. pisum. Phylogenetic analysis showed that the trees for the CP and the C-terminal half of the RTD sequences contained clusters of isolates of the same symptom type, whereas the tree for the N-terminal half of the RTD contained clusters of isolates of the same aphid vector type. These results agreed with our previous data of the complete nucleotide sequences of four SbDV isolates, and strongly indicated a close relationship between the N-terminal half of the RTD amino acid sequences and aphid transmission specificity of SbDV.