First Report of Group 16SrXII Phytoplasma Associated with Papaya Yellows in Taiwan.

Phytoplasmas have been reported to cause various disorders in papaya (Carica papaya L.), including dieback, mosaic, and yellow crinkle in Australia, Nivun Haamir dieback in Israel, and bunchy top-like disease in Cuba (1). Papaya is an economically important crop in Taiwan, and therefore, is monitored for viral infections. In 2005, papaya plants showing chlorosis, yellows and shriveling of leaves, dieback and lateral growth of branches, bending of apical branches, latexosis of fruits, and brown necrosis in phloem tissues were observed in southern Taiwan. Examination by an electron microscope revealed the presence of pleomorphic phytoplasma cells in sieve tubes of the phloem of petioles and leaf veins of diseased plants. Total DNA was extracted individually from at least three diseased plants at each location with a commercial DNA preparation kit (Axygen Scientific, Union City, CA) and used for amplification of the phytoplasma 16S rRNA gene in PCR with universal primer pairs P1 and Tint (3). The full-length 16S rRNA gene has been amplified and cloned. Sequence analysis revealed that the fragment was 1,581 bp long (GenBank Accession No. AJ919994) and shared 99.6% sequence identity with that of the 'Candidatus Phytoplasma solani' reference strain (GenBank Accession No. AF248959). A virtual restriction fragment length polymorphism analysis of the 16S rDNA sequence amplified from the R16F2n/R16R2 primers (2) was performed with iPhyClassifier (4) and pDRAW32. In silico restriction analysis identified the studied papaya phytoplasma as a subgroup 16SrXII-A strain. The sequence had 97 to 98% sequence identity with papaya phytoplasmas of the 16SrXII group in Australia (GenBank Accession No. Y10095), Israel (GenBank Accession No. AY903951), and Cuba (GenBank Accession No. AY725234). The disease incidence was 30 to 35% during the 2006 to 2010 growing seasons, and field surveys indicated that the disease has spread to central Taiwan with sporadic occurrence in recent years. To our knowledge, this is the first report of phytoplasma associated with papaya yellows in Taiwan. References: (1) Y. Arocha et al. Int. J. Syst. Evol. Microbiol. 55:2451, 2005. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of Turnip ringspot virus in Field Mustard (Brassica chinensis) in Taiwan.

Crucifer crops (Brassica spp.) are important winter vegetables in Taiwan. Five viruses, including Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV), Radish mosaic virus (RaMV), Beet western yellows virus (BWYV), and Cauliflower mosaic virus (CaMV), have been detected in a range of domestic-grown crucifers during past decades (1). Field mustard plants (Brassica chinensis) showing mosaic in the leaves were collected in the ChiaYi area in December 2007. Spherical virus-like particles, approximately 30 nm in diameter, were readily observed in crude sap of symptomatic plants. Tests by ELISA failed to detect any of the aforementioned viruses. A spherical agent was isolated through mechanical inoculation onto Chenopodium quinoa, and a virus culture was established and inoculated mechanically back to the original host as well as other crucifers. Systemic mosaic appeared on inoculated B. campestris, B. chinensis, and B. juncea, whereas ringspots appeared on inoculated leaves of B. oleracea. Total RNA was extracted from symptomatic leaves and used for reverse transcription (RT)-PCR amplification using degenerate primers for comoviruses (2). Other successive fragments of RNAs 1 and 2 were amplified by specific or degenerate primers designed on the basis of sequences of published Turnip ringspot virus (TuRSV). The RNA 1 (GenBank Accession No. GU968732) and RNA 2 (No. GU968731) of the isolated virus consisted of 6,076 and 3,960 nucleotides, respectively. The number of nucleotides and the arrangement of open reading frames on both RNA 1 and RNA 2 were similar to those of comoviruses. Sequence analysis revealed that the nucleotide sequences of RNA 1 and RNA 2 shared 54.2 to 82.5% and 50.2 to 79.3% similarities, respectively, to those of comoviruses and were most similar to Turnip ringspot virus. The deduced peptides of large and small coat proteins (LCP and SCP) contain 375 amino acids (41.2 kDa) and 251 amino acids (28.5 kDa), respectively. The deduced amino acid sequences of RNA-dependent RNA polymerase (RdRp), LCP, and SCP share 92.0 to 94.5%, 93.1 to 93.3% and 87.3 to 89.6% similarity, respectively, to those of published TuRSV isolates, i.e., -B (GenBank Accession No. GQ222382), -M12 (No. FJ516746), and -Toledo (No. FJ712027) indicating that the newly isolated virus from field mustard in Taiwan is an isolate of TuRSV, hence TuRSV-TW. Comparison of LCP and SCP between current TuRSV-TW and Radish mosaic virus (RaMV; GenBank Accession No. AB295644) showed 74% similarity, which is below the species demarcation level of 75% (3), indicating its discrimination from RaMV. To our knowledge, this is the first report of the occurrence of TuRSV in Taiwan and in the subtropics. References: (1) T. H. Chen et al. Plant Pathol. Bull. 9:39, 2000. (2) V. Maliogka et al. J. Phytopathol. 152:404, 2004. (3) K. Petrzik and I. Koloniuk. Virus Genes 40:290, 2010.

Potential threat of a new pathotype of Papaya leaf distortion mosaic virus infecting transgenic papaya resistant to Papaya ringspot virus.

A virus identified as a new pathotype of Papaya leaf distortion mosaic virus (PLDMV, P-TW-WF) was isolated from diseased papaya in an isolated test-field in central Taiwan, where transgenic papaya lines resistant to Papaya ringspot virus (PRSV) were evaluated. The infected plants displayed severe mosaic, distortion and shoe-stringing on leaves; stunting in apex; and water-soaking on petioles and stems. This virus, which did not react in enzyme-linked immunosorbent assay with the antiserum to the PRSV coat protein, infected only papaya, but not the other 18 plant species tested. Virions studied under electron microscope exhibited morphology and dimensions of potyvirus particles. Reverse transcription-polymerase chain reaction conducted using potyvirus-specific primers generated a 1,927-nucleotide product corresponding to the 3' region of a potyvirus, showing high sequence identity to the CP gene and 3' noncoding region of PLDMV. Search for similar isolates with the antiserum against CP of P-TW-WF revealed scattered occurrence of PLDMV in Taiwan. Phylogenetic analysis of PLDMV isolates of Taiwan and Japan indicated that the Taiwan isolates belong to a separate genetic cluster. Since all the Taiwan isolates infected only papaya, unlike the cucurbit-infecting Japanese P type isolates, the Taiwan isolates are considered a new pathotype of PLDMV. Susceptibility of all our PRSV-resistant transgenic papaya lines to PLDMV indicates that the virus is an emerging threat for the application of PRSV-resistant transgenic papaya in Taiwan and elsewhere.

The primary immunity determinant in modulating the lysogenic immunity of the filamentous bacteriophage cf.

Bacteriophage cf is the first single-stranded DNA phage that has been shown to set up a stable lysogenic state with its genome integrated into the host chromosome. From the isolation and characterization of a virulent mutant, cf-tv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage. The mutation in cf-tv2 enables the phage to produce plaques on lawns of cf lysogenic cells. The mutation was defined as a 49-nucleotide deletion located in a 0.59 kb NcoI/KpnI fragment of cf replicative form DNA. Two messages, cM1 and cM2, transcribed from the immunity region of wild-type cf but in opposite directions, were detected. In cf-tv2, the 49-nucleotide deletion abolishes cM2 transcription. The primer extension assay suggests a possible RNA-RNA interaction directed by base-pairing of the cM1 and cM2 RNAs. A frameshift mutation of the open reading frame ORF 165, encoded by cM2, resulted in a 10(5) plating efficiency on the cf lysogen. These observations suggest that both RNA-RNA interaction and repressor protein inhibition are involved in the mechanism of cf immunity. A model is proposed for the regulation of cf immunity.

Complete nucleotide sequence and genetic organization of papaya ringspot virus RNA.

The complete nucleotide sequence of the RNA genome of papaya ringspot virus (PRSV) was determined from four overlapping cDNA clones and by direct sequencing of viral RNA. The genomic RNA is 10326 nucleotides in length, excluding the poly(A) tract, and contains one large open reading frame that starts at nucleotide positions 86 to 88 and ends at positions 10118 to 10120, encoding a polyprotein of 3344 amino acids. The highly conserved sequence AAAUAAAANANCUCAACACAACAUA at the 5' end of the RNA of PRSV and those of the other five reported potyviruses shows 80% similarity, suggesting that this region may play a common important role for potyvirus replication. Two cleavage sites of the polyprotein were determined by amino acid sequencing of the N termini of helper component (HC-Pro, amorphous inclusion) and cylindrical inclusion (CI) proteins. Other cleavage sites were predicted by analogy with the other potyviruses. The genetic organization of PRSV is similar to that of the other potyviruses except that the first protein processed from the N terminus of the polyprotein (NT protein) has an M(r) of 63K, 18K to 34K larger than those of the other potyviruses. The cleavage site for liberating the N terminus of the HC-Pro protein was found at the same location down-stream from the consensus sequence FI(V)VRG as that reported for tobacco vein mottling virus. The NT protein of potyviruses is the most variable and may be considered important for identification of individual potyviruses. The most conserved protein of potyviruses appears to be the NIb protein, the putative polymerase for the replication of the potyviral RNA. The genetic organization of PRSV RNA is tentatively proposed to be VPg-5' leader-63K NT-52K HC-Pro-46K-72K CI-6K-48K NIa-59K NIb-35K coat protein-3' non-coding region-poly(A) tract.

Identification and nucleotide sequence analysis of an open reading frame involved in high-frequency conversion of turbid to clear plaque mutants of filamentous phage Cf1t.

Clear plaque mutants (Cf1c) isolated from the temperate filamentous phage Cf1t occurred at a frequency of approximately 10(-3). The pahge yield from Cf1c-infected cells was higher than that from Cf1t-infected cells. Results of spot complementation tests implied that the turbid plaque phenotype is dominant. DNA fragment substitution studies indicated that a NcoI/KpnI fragment of 591 bp was responsible for the determination of plaque turbidity. Sequence data from four Cf1c isolates revealed base pair alterations and a deletion located in the upstream region of an open reading frame (ORFII) which might encode a 18.2-kDa protein. When the ORFII in Cf1t was disrupted by a frameshift mutation, this recombinant phage formed clear plaques. These observations suggest that ORFII may participate in the formation of turbid plaques. ORFII does not show significant homology with the sequence of f1 gpII, gpV, or other known phage proteins.