H. R. Chen

Complete nucleotide sequence and genome organization of a Chinese isolate of tobacco bushy top virus

Summary.u2002The complete nucleotide sequence of a Chinese isolate of tobacco bushy top virus (TBTV), designated TBTV-Ch, was determined from cDNA generated from double-stranded RNA extracted from diseased tobacco. The genome is 4152 nucleotides (nt) in size, contains four putative open reading frames (ORFs) and untranslated regions of 10u2009nt and 645u2009nt at the 5′ and 3′ ends, respectively. In genome organization and in the amino acid sequence of its potential products, the RNA of TBTV-Ch is similar to other umbraviruses sequenced to date. The results suggested that TBTV should be regarded as a definitive species of the genus Umbravirus.

First report of pomegranate wilt caused by Ceratocystis fimbriata in Yunnan, China.

Pomegranate (Punica granatum Linn.) is an important fruit crop in Yunnan Province, China. Recently, older pomegranate bushes in Mengzi County, Yunnan began dying. Initial symptoms were yellowing and wilting of leaves on one to several branches, followed by sudden death of the bush within 3 to 4 weeks. Roots of diseased bushes appeared brown to black, and irregularly shaped lesions were observed when the bark was removed. A species of Ceratocystis was consistently isolated from discolored roots, stem, and branch tissues from wilted bushes on potato dextrose agar (PDA) and was identified as Ceratocystis fimbriata Ellis & Halst, based on the morphology of perithecia, ascospores, conidia, and conidiophores. Perithecia were black with a globose base (130 to 300 μm) and a long neck (450 to 800 μm). Ascospores exuded from the apex of the perithecium neck in a long coil and were small, hyaline, and hat-shaped (3.8 to 5.0 μm long × 2.3 to 4.0 μm wide). Conidiophores were septate and hyaline to dark greenish brown. Hyaline conidia, 8 to 17 μm long × 6 to 15 μm wide, were usually produced in chains of 10 or more. Thick-walled endoconidia were globose to oval, olive brown, and 8 to 20 μm in diameter. Because of the increasing occurrence of the disease, surveys of the main pomegranate production areas in Mengzi County, including Xinan, Duofale, Caoba, Hongzhai, and Shilipu townships, were conducted from 10 to 20 August 2002. The disease was detected in 17 of 50 plantings surveyed. Disease was more severe in older plantings than in younger plantings. Disease incidence was 1% in 1- to 5-year-old bushes, 3.6% in 6- to 10-year-old bushes, and 6% in bushes more than 10 years old. Scolytid beetles were occasionally found on bushes, but we were unable to isolate the fungus from them as has been reported (1). Inoculations with an isolate of C. fimbrata were made by inserting mycelium with perithecia from 12-day-old cultures growing on PDA into root wounds made with a sterile scalpel on five pomegranate plants and then covering the wounds with Parafilm. Sterile medium was placed in an equal number of wounded bushes to serve as controls. Fourteen days later, symptoms began to appear in two bushes, and 5 days later, all bushes exhibited symptoms. No symptoms were observed on control bushes. The first visible symptom was a small area of blackened tissue near the point of inoculation. Lesions expanded slowly, but they expanded more rapidly upward than downward. The fungus was reisolated on PDA from roots of all artificially inoculated bushes. C. fimbriata has been previously reported as the cause of pomegranate wilt in India (2); however, to our knowledge, this is the first report of C. fimbriata on pomegranate in China. Because environmental conditions which favor the pathogen (temperatures ranging from 18 to 30°C and frequent rains) typically occur in many areas during late spring and summer, the disease has the potential to seriously impact pomegranate production in China. References: (1) Y. M. Somasekhara. Plant Dis. 83:400, 1999. (2) Y. M. Somasekhara, et al. Res. Crops 1(1):63, 2000.

First Report of Tobacco Bushy Top Disease in China

In 1993, a severe epidemic of a new disease of flue-cured tobacco (Nicotiana tabacum) occurred in western Yunnan Province, Peoples Republic of China. Since then, over 40,000 ha of tobacco have been affected, with an average incidence of ≈15%. Infected plants were stunted, and leaves showed symptoms of vein distortion, vein clearing, mottling, and rounding. Axillary buds sprouted from the main stem of infected plants early and formed lateral shoots, on which other shoots were produced. As a result, the infected plants presented the characteristic bushy appearance. Aphid- (Myzus persicae (Sulzer)) and sap-transmission experiments were conducted. In aphid-transmission tests, after an acquisition feeding period of 24 h on diseased tobacco, the shortest test feeding that resulted in infection was 5 min. The causal agent(s) was readily sap-transmissible, but it could not be transmitted from sap-inoculated plants to healthy plants by aphids. Thirty-two species of plants in eleven families were tested by sap-inoculation for infectivity to alternative hosts by the casual agent(s). All the species of Nicotiana tested were infected. All the hosts were restricted to the solanaceae. The symptoms, transmission, and host range of this disease were identical to those of tobacco bushy top disease in Zimbabwe (1). Using umbravirus-specific primers (3), a 550-bp DNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from diseased tobacco, whose sequence (GenBank Accession No. AF402620) indicated the occurrence of an umbravirus. The results coincided with the taxonomic status of Tobacco bushy top virus, one of the causal agents of tobacco bushy top disease (1), a tentative species of the genus Umbravirus (2). To our knowledge, this is the first report of tobacco bushy top disease in China and the first report of a large outbreak of the disease outside sub-Saharan Africa. References: (1) L. F. Gates. Ann. Appl. Biol. 50:169, 1962. (2) F. A. Murphy. et al. Page 388 in: Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses, Page 388, 1995. (3) P. Vercruysse et al. J. Virol. Methods 88:153, 2000.

Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR.

Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay was developed for simultaneous detection of the four causal agents of the disease. Four pairs of specific primers based on the conserved regions of each of the four disease agents were used in the one-tube RT-PCR. The RT-PCR products consisted of fragments of 1049 base pairs (bp) for TBTV, 792bp for TVDVaRNA, 598bp for Sat-TBTV and 357bp for TVDV, and their origins were confirmed by sequencing. Primer concentrations and cycling condition were optimized for the multiplex RT-PCR. The detection limit of the assay was up to 10(-4) dilution. The assay was evaluated using tobacco plants infected naturally with one to four target viruses, transmission vector of aphids and field samples collected from Yunnan, Hunan, and Guizhou province, China. The results show that the multiplex RT-PCR is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in tobacco and aphid. This assay will be useful for virus surveys when large numbers of samples are tested.

First report of a natural infection of Opuntia sp. by a 'Candidatus Phytoplasma asteris'-related phytoplasma in China.

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a Candidatus Phytoplasma asteris-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) E. Choueiri et al. Plant Dis. 89:1129, 2005. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (4) N. Leyva-Lopez et al. Phytopathology (Abstr.) 89(suppl):S45, 1999.

Molecular characterization of a new macluravirus from yam in Yunnan, China.

Yams (Dioscorea spp.) are important in China as vegetables and as sources of traditional herbal medicine. They suffer from various viral diseases, but there has only been one small study of them in China [6]. We have investigated the viruses associated with diseased yams at two farms 5 km apart in Yongsheng county, Yunnan province. Samples 1–3 from D. zingbierensis had a combination of vein clearing, veinal necrosis and chlorotic symptoms on the expanded leaves. Samples 1 and 2 were from the same farm. Sample 4 from D. parviflora (same site as sample 3) had systemic chlorosis, mottling and mosaic symptoms that also affected the newly emerging leaves. Virus particles and inclusion bodies typical of members of the family Potyviridae were observed in infected leaves. Particles were purified [3], RNA was extracted and the 30-terminal portion of the viral genome was then amplified by RT-PCR by standard methods using universal Potyviridae primers [4]. Products were separated, purified and cloned (into pMD19-T; TaKaRa), and three independent clones were auto-sequenced in both directions using an ABI PRISM 3770 DNA Sequencer. Sequence alignments and phylogenetic analysis were done using MEGA 4.0 [7]. Fragments of 1,742–1,748 nucleotides in length were amplified and sequenced from each sample. Isolates 1, 3 and 4 were almost identical to one another (99.4–99.5% nucleotide identities) while isolate 2 had 78.2% identity to the others. The sequences consisted of an incomplete open reading frame with a stop codon at position 1531–1533 followed by a 30-UTR of 209 nucleotides (215 in sample 2). In comparisons with published sequences, the nearest matches were those for Chinese yam necrotic mosaic virus (ChYNMV, genus Macluravirus), with about 69% nt and 72–73% aa identity. Comparisons with all other macluravirus sequences suggested that the partial ORF of 510 amino acid residues in the new sequences consisted of 222 aa at the C-terminus of the NIb and a coat protein (CP) of 288 aa (32.3 kDa) with a cleavage site of INLQ/M (IDLQ/ M in sample 2). The LQ/M cleavage is typical of members of the genus Macluravirus [1]. The partial NIb gene contained the consensus motifs (T/S)GX3-TX3-N(T/S) and GDD of the RNA-dependent RNA polymerase. The N-terminus of the CP did not contain a DAG (or similar) motif that is known to be associated with aphid transmission in the genus Potyvirus. Molecular criteria for discriminating between species in the family Potyviridae indicate a threshold of 76% (nt) or 82% (aa) identity [2]. Based on these criteria, the new yam sequences belong to a single new species in the genus Macluravirus. These conclusions were confirmed by phylogenetic analysis (Fig. 1). Although isolate 2 is rather different to the others at the molecular level, it was one of the three from D. zingbierensis that had similar symptoms and is not sufficiently different to justify placement in a further new species. Interestingly, there is a similar range of differences between isolates of Cardamom mosaic virus J.-G. Wang X.-J. Zou H.-R. Chen (&) The Phytopathology Laboratory of Yunnan Province, Yunnan Agricultural University, 650201 Kunming, People’s Republic of China e-mail: hrchen44@yahoo.cn

First Report of Botryosphaeria dothidea Causing Canker and Shoot Blight of Eucalyptus in China

Eucalyptus spp. are grown on approximately 2 million ha in China and is the most important genus in forest plantations worldwide. An outbreak of cankers and shoot blight was observed for the first time on Eucalyptus grandis in China during May of 2008. Symptoms included dieback of shoots and branches, lesions and canker formation on the stems, and clear or yellow-to-red exudates on stems and branches. Isolations were made from affected trees in Shilin County from May to July of 2008. Diseased samples were plated on potato dextrose agar (PDA) and incubated at 26°C. Fungal isolates developed copious, white, aerial mycelium that became dark gray after 5 to 6 days and formed black pycnidia after 14 days. Conidia were hyaline, aseptate, thin walled, fusiform, and measured 19 to 28 × 4 to 6 μm. Ascospores were hyaline, aseptate, and widest from the middle to upper third (17 to 28 × 6 to 13 μm). Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2) with primers ITS1 and ITS4. BLAST searches showed 99 to 100% identity with Botryosphaeria dothidea isolates from GenBank (Accession Nos. FJ358703 and EU080916). Representative sequences of B. dothidea from eucalyptus from China were deposited into GenBank (Accession Nos. FJ517657 and FJ517658). On the basis of morphological and molecular results, the fungus isolated from diseased eucalyptus wood was confirmed to be B. dothidea. Pathogenicity tests were conducted by stem inoculation of 10-month-old E. grandis seedlings. Two experiments were conducted using two inoculation techniques. In the first experiment, 2-mm-diameter actively growing mycelium plugs of B. dothidea were applied to 2-mm-long bark wounds on the middle point of the stems, and control seedlings were inoculated with sterile PDA plugs in a similar fashion as above. Inoculated and control seedlings were inoculated in a greenhouse and watered as needed. In the second experiment, segments of branches (averaging 18 mm in diameter and 30 cm long) were inoculated with 5-mm-diameter plugs of actively growing mycelium. Control segments of branches were inoculated as previously described. The branches were incubated at 26°C in moist chambers. There were five replicate seedlings per inoculation technique. After 20 days, all E. grandis seedlings showed leaf wilting, Dark, vascular stem tissue was observed. Symptoms were more abundant on the segment of branches. After 6 days, vascular necroses that developed on the inoculated plants were 5.2 ± 1.2 cm. B. dothidea was reisolated from all inoculated symptomatic tissue; no symptoms were visible in the control seedlings and no fungus was isolated from them. These results confirm previous reports of B. dothidea causing canker and dieback symptoms of Eucalyptus species in Australia (2), the United States (1), and South Africa (3). To our knowledge, this is the first report of B. dothidea causing canker disease on eucalyptus in China. References: (1) E. L. Barnard et al. Plant Dis. 71:358, 1987. (2) E. M. Davison and C. S. Tay. Plant Dis. 67:1285, 1983. (3) H. Smith et al. Plant Pathol. 43:1031, 1994.

Complete genome sequence of yam chlorotic necrosis virus, a novel macluravirus infecting yam

The complete genome sequence of a novel member of the genus Macluravirus was determined from yam plants with chlorotic and necrotic symptoms in China. The genomic RNA consists of 8,261 nucleotides (nt) excluding the 3’-terminal poly(A) tail, containing one long open reading frame (ORF) encoding a large putative polyprotein of 2,627 amino acids. Its genomic structure is typical of macluraviruses, which lack the P1 protein, N-terminal HC-Pro, and D-A-G motif for aphid transmission that are found in potyviruses. The virus shares 56.3-63.8% sequence identity at the genome sequence level and 49.7-63.9% at the polyprotein sequence level with other members of the genus Macluravirus. Phylogenetic analysis based on the complete polyprotein sequence of representative members of the family Potyviridae clearly places the virus within the genus Macluravirus. These results suggest that the virus, tentatively named “yam chlorotic necrosis virus” (YCNV), should be considered a member of a novel species in the genus Macluravirus.