A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-95-0166

A one-step reverse transcription-polymerase chain reaction system for the simultaneous detection and identification of multiple tospovirus infections.

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes.

The W8 isolate of the phytopathogenic fungus, Rosellinia necatrix that causes white root rot, contained three segments of double-stranded (ds) RNA, namely L1, L2 and M. Purified viral particles of about 25 nm in diameter contained an RNA segment with almost the same mobility as M-dsRNA, but the band was sensitive to S1 nuclease. Molecular analysis revealed that M-dsRNA consisted of two (RNA 1 and RNA 2) similarly sized species of 2299 and 2279 bp excluding an interrupted poly (A or U) tail of 16-51 bp. The predicted largest open reading frame in RNA 1 and RNA 2 was similar to those of RNA dependent RNA polymerase (RdRp) and coat protein (CP), respectively, encoded by the family Partitiviridae. The non-coding regions (NCR) of the two segments were similar (approximately 70% base identity) at the 5' end, but different at the 3' end. The NCR at the 3' end contained adenosine-uracil rich elements (AREs) in both segments. Northern analyses revealed RNA 1 and RNA 2 in mycelial and viral particle fractions. We coined the name Rosellinia necatrix partitivirus 1-W8 (RnPV1-W8) for M-dsRNA based on viral particle morphology and sequence information.

Molecular characterization of a partitivirus from the plant pathogenic ascomycete Rosellinia necatrix

Several field isolates of Pepper mild mottle virus (PMMoV) were obtained from Capsicum annuum (L

 3
 
/ + ) that exhibited systemic mosaic or systemic necrosis The deduced amino acid sequences of the coat protein (CP) of these isolates differed from those of previously reported PMMoV strains that are able to overcome L

 3
 gene-mediated resistance CP mutants having these amino acid changes were constructed using cDNA clones pTPWl and pTPC4350 These clones produce in vitro transcripts TPW1, which induces a resistance response resulting in local infection, and TPC4350, which causes no response on inoculated leaves resulting in systemic infection in the Capsicum plants carrying the L

 3
 gene (L

 3
 
/L

 3
 
) These CP mutants were tested for infection and symptomatology in Capsicum spp plants (L

 3
 
/L

 3 and L

 3
 
/ + ) An A/S (from serine to alanine) change at position 81 in the CP of TPW1 conferred a resistance-breaking activity in the L

 3
 
/L

 3
 plants, whereas an S/A change at position 7 in the CP of either TPW1 or TC4350 conferred resistance-inducing activities These results suggest that amino acid changes of A/S at position 81 and S/A at position 7 in CP had inverse effects on the L

 3
 gene-mediated resistance These and other amino acid changes in PMMoV CP are also discussed in relation to the dosage effects of the L

 3
 gene on virus resistance

Amino Acid Changes in Pepper mild mottle virus Coat Protein That Affect L3 Gene-mediated Resistance in Pepper'

The complete nucleotide sequence of an attenuated Pepper mild mottle virus (PMMoV C-1421) RNA genome has been determined. There were two differences from the type isolate in Japan (PMMoV-J). The mutations were located in the middle of the 126-kDa protein (126 K) gene; one mutation influenced amino acid substitution at 649th Val to Ala (V649A), and the other was silent. The analyses using the reverse genetic system of PMMoV-J revealed that symptom attenuation on pepper related to V649A. Accumulations of 126 K and coat protein (CP) in V649A mutant-infected pepper were lower than those of PMMoV-J in immunoblotting. These results suggest that V649A substitution in 126 K affects the accumulation of 126 K leading to a limitation of CP accumulation.

A single amino acid substitution in 126-kDa protein of Pepper mild mottle virus associates with symptom attenuation in pepper; the complete nucleotide sequence of an attenuated strain, C-1421.

新哉津田

Papers

Nucleotide Sequence of the Japanese Isolate of Pepper Mild Mottle Tobamovirus (TMV-P) RNA.

Detection of Tomato Spotted Wilt Virus S RNA in Individual Thrips by Reverse Transcription and Polymerase Chain Reaction.

Purification of Tomato Spotted Wilt Virus Isolated from Tobacco Plants in Morioka, Japan.

The Presence of Three Pairs of Possibly Complementary RNA Species in Isolated Nucleocapsid Material of Tomato Spotted Wilt Virus.

Simultaneous Diagnosis for Plants Infected with Multiple Viruses Employing Rapid Immunofilter Paper Assay(RIPA) with Two-Step Method; Multi-RIPA.

新哉 津田