Hiroyuki Ieki

Proposal for rejection of Agrobacterium tumefaciens and revised descriptions for the genus Agrobacterium and for Agrobacterium radiobacter and Agrobacterium rhizogenes.

The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined. These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria. However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated. In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated. The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars. We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter (for the biovar 1 strains) and Agrobacterium rhizogenes (for the biovar 2 strains) are proposed. The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction.

We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) to detect six citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV) and Citrus viroid OS (CVd-OS) and Apple stem grooving virus (ASGV, synonym: Citrus tatter leaf virus (CTLV)) from citrus plants. The multiplex RT-PCR was also designed to distinguish CVd-I-LSS (a distinct variant of CBLVd) from CBLVd. By the multiplex RT-PCR, one to eight fragments specific to the pathogens were simultaneously amplified from one sample and identified by their specific molecular sizes in 6% polyacrylamide gel electrophoresis. The results of the multiplex RT-PCR were consistent with those of other diagnoses, such as uniplex RT-PCR, to detect each of the pathogens. The multiplex RT-PCR provides a simple and rapid method for detecting various viroids and ASGV in citrus plants, which will help diagnose many citrus plants at a time.

Nucleotide Sequence of Citrus Tristeza Virus Seedling Yellows Isolate

The complete nucleotide sequence of a seedling-yellows-inducing isolate NUagA of Citrus tristeza virus (CTV) was determined. It consisted of 19302 nucleotides and contained 12 open reading frames (ORF) organized identically to those of previously sequenced isolates. This genome is the largest among the CTV genome sequenced so far ; it is 6 nucleotides (nt), 76 nt, 43 nt, and 53 nt longer than that of T36 (quick decline, Florida), VT (seedling yellows, Israel), T385 (mild, Spain), and SY568 (stem pitting, California), respectively. Sequence comparison of NUagA and the other isolates revealed approximately 90% identities throughout the 3′ half of the genome. The 5′ half of the genome was only about 70% identical to that of T36 but still high at about 90% to those of VT, SY568, and T385. Comparison of amino acid sequences on ORF1a encoding polyproteins, the most variable region, reflects the CTV isolate relationship ; NUagA is closely related to VT, SY568, and T385, but distantly related to T36.

Comparison of 16S rDNA and 16S/23S intergenic region sequences among citrus greening organisms in Asia

Polymerase chain reaction was used to amplify and sequence the 16S ribosomal RNA gene (rDNA) and 16S/23S intergenic region of several isolates of citrus greening organism (GO) from Japan, the Philippines, Indonesia, and Thailand. The sequences of 16S rDNA were identical among all the isolates studied, very similar to the published sequences of Thai (99.4 to 100% identity), Nepalese (100% identity), and Indian (98.8% identity) strains, and less similar to an African strain (97.5% identity). The sequences of the intergenic region between 16S and 23S rDNA were also identical among the isolates examined as well as the reported Nepalese and Thai isolates. They were close to the sequences of reported strains of India and China (99.2%) and apart from those of the African strain (85.5%). These results suggested that some isolates of GO from Japan, the Philippines, Indonesia, Thailand, and Nepal constitute one strain, which is similar to Indian and Chinese strains and distinct from the African strain.

Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region

SummaryNucleotide sequences of group I citrus viroids Ia (CVd-Ia) and citrus bent leaf viroid (CBLVd, formerly designated CVd-Ib) isolated from citrus plants in Japan, the Philippines and China have been determined. Citrus samples in Japan and the Philippines contained CVd-Ia, which consists of 328 nucleotides(nt). Although 10 nt longer than the type CBLVd-225A in Israel they share 94% identity in overall nucleotide sequence. The Philippines sample also contained a 329-nt long CVd-Ia sequence variant, in which one base insertion and three substitutions were observed. A citrus in China contained CBLVd, which consists of 318 nt and shares 98% identity to CBLVd-225A. CVd-Ia was clearly separated from CBLVd by two 5-nt insertions located in upper (5′-AGCUG-3′) and the lower (5′-CUUCU-3′) strand of the right terminal region (which is also designated T2 domain) in rod-like secondary structure. Since both of the additional 5-nt sequences are similar to the adjacent sequences (5′-AGUUG-3′ and 5′-CUUCU-3′), we hypothesize that CVd-Ia is a derivative of CBLVd caused by partial sequence duplications and substitutions taking place in the right terminal region.

The nucleotide sequence of the coat protein genes of satsuma dwarf virus and navel orange infectious mottling virus

SummaryThe sequence of the 3′-terminal 4320 and 2409 nucleotides were determined for RNAff2 of satsuma dwarf virus (SDV) and navel infectious mottling virus (NIMV). Both sequences contained a part of a long open reading frame which encodes larger and smaller coat proteins (CPs) at the 3′ terminus followed by a 3′ non-coding region upstream of a poly(A) tail. Amino acid sequence identity for larger and smaller CPs ranged 81–84% and 68–78%, respectively, among SDV, NIMV and the previously sequenced citrus mosaic virus (CiMV). No significant sequence similarity was found between the CPs of SDV or NIMV and those of the como-, nepo- or other viruses. The nucleotide sequence identity of the 3′ non-coding region of RNAff2 were 68%–78% among SDV, CiMV and NIMV. These results suggest that SDV, CiMV and NIMV are distinct, though related, viruses. They may be assigned as members of the new genus, which is close to the genera of Comovirus and Nepovirus.

A new virus related to Satsuma dwarf virus: the nucleotide sequence of the 3 -terminal regions of Hyuganatsu virus RNAs 1 and 2 ∗

Summary.The sequences of the 3′-terminal 1306 and 2160 nucleotides of RNAs 1 and 2 of a virus serologically related to Satsuma dwarf virus (SDV) from Hyuganatsu (Citrus tamurana Hort. ex Tan.) were determined, respectively. We found that the partial RNA-dependent RNA polymerase region in RNA1 and the coat proteins (CPs) region in RNA2 of the virus tentatively named Hyuganatsu virus (HV) have 78.3–84.0% and 76.9–80.7% amino acid sequence identities to those of known SDV-related viruses (SDV-RVs), i.e., SDV, Citrus mosaic virus, and Navel orange infectious mottling virus. Sequence analyses show that HV is classifiable as a new SDV-RV species.

Reduction of kiwifruit ripe rot by accelerated chemical ripening at low temperatures

Abstract When kiwifruit (Actinidia deliciosa (A. Chev.) CF. Liang et A.R. Ferguson) stored at low temperatures (2-5°C) for several months were treated by gases evolved from chemicals newly developed for promoting ripening (Kanjuku Pack (KP) produced by Shiraishi Calcium Co. Ltd.), fruits quickly ripened at 15°C for c. 8 days, and the occur-rence of ripe rot was significantly suppressed. Hyphal growth of the causal fungi, Phomopsis sp. and Botryosphaeria sp., on potato dextrose agar medium was also suppressed by treatment with KP. A KP evolves ethylene, ethyl alcohol,acetaldeyde,hydrogen, and carbon monoxide. Acetaldehyde gas actively suppressed hyphal growth of both fungi, and also ethyl alcohol gas gave slight suppression.