Joe Tang

First report of Narcissus degeneration virus, Narcissus late season yellows virus, and Narcissus symptomless virus on Narcissus in New Zealand.

In September 2008, Narcissus plants originating from commercial nurseries in Taranaki (TK) in New Zealands North Island and Canterbury (CB) in the South Island were received showing leaf mottling, flower distortion, and color break. The CB plant also showed stunting. Filamentous virus particles (700 to 900 nm long) were seen in crude sap of both plants with a transmission electron microscope. Total RNA was isolated from the leaves of both plants with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA), and cDNA was synthesized by Superscript III (Invitrogen, Carlsbad, CA). cDNA was used in PCR to test for viruses in the following genera: Allexivirus, Carlavirus, Cucumovirus, Nepovirus A and B, Potyvirus, Potexvirus, Tospovirus, and Tobravirus. Both plants tested positive for potyvirus using generic potyvirus primers (3). Amplicons from both plants were directly sequenced. The forward and reverse sequence from the CB plant matched sequences in the GenBank database for Narcissus late season yellows virus (NLSYV) and Narcissus degeneration virus (NDV), respectively. The potyvirus amplicon from the CB plant was cloned and sequenced. Sequence from independent clones was obtained for NLYSV only (No. FJ546721), and this sequence showed 97% nucleotide identity to NLYSV No. EU887015. The CB plant was tested with a second set of generic potyvirus primers using forward (PV1SP6) (2) and reverse primers (U335) (1). BLASTN analysis of the sequence obtained from independent clones (No. FJ543718) matched sequence for NDV only (97% nucleotide identity to No. AM182028). BLASTN analysis of the potyvirus obtained for the TK plant (No. FJ546720) showed 97% nucleotide identity to NLSYV (No. EU887015). The TK plant also tested positive for a carlavirus using commercial primers (Agdia, Elkhart, IN) and unpublished generic carlavirus primers (A. Blowers, personal communication). Amplicons from both PCRs were cloned and sequenced. BLASTN analysis of both sequences (Nos. FJ546719 and GQ205442) showed 94% nucleotide identity to Narcissus symptomless virus (NSV) No. AM182569. Both plants were also tested for NLSYV, Narcissus virus Q, Narcissus latent virus, and Narcissus yellow stripe virus by indirect ELISA (Neogen, Lansing, MI). Results confirmed the presence of NLSYV in both plants but the plants were negative for the other viruses. NLSYV has been detected previously from Narcissus pseudonarcissus L. (daffodil) (D. Hunter, personal communication); however, to our knowledge, this is the first official report of NDV, NLSYV, and NSV in New Zealand. Since both plants tested negative for several other viruses by PCR and ELISA, this would suggest that the symptoms observed may have been caused by NSV, NLSYV, NDV, or as a result of a mixed infection. However, symptoms were not confirmed using Kochs postulate. NSV has been reported in the literature as symptomless. NLYSV has been reported to be a possible cause of leaf chlorosis and striping and NDV has been associated with chlorotic leaf striping in N. tazetta plants (4). Since Narcissus is an important flower crop for domestic production in New Zealand, the reduction in flower quality observed when these viruses are present may be of economic significance in commercial nurseries. References: (1) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (2) A. M. Mackenzie et al. Arch Virol. 143:903, 1998. (3) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000. (4) W. P. Mowat et al. Ann. Appl. Biol. 113:531, 1988.

A review of the plant virus, viroid, liberibacter and phytoplasma records for New Zealand

A complete review of the records of plant virus, viroid, liberibacter and phytoplasma in New Zealand has found evidence for 220 viruses, seven viroids, two liberibacters and two phytoplasmas. Of these, 80 viruses, one viroid and two species of liberibacter have been reported as new to New Zealand since the last review in 2006. Ten viruses and two viroids, which were previously placed in the unconfirmed category, have now been confirmed. Based on insufficient evidence, 25 virus, three viroid, three mollicute and 36 disease records are considered unconfirmed.

Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution

Tomato spotted wilt virus (TSWV) is an internationally significant pathogen with a wide host range, vectored by thrips. We have studied the sequence variation and evolutionary mechanisms at play in parts of the L, M and S subgenomes of 23 New Zealand TSWV isolates collected between 1992 and 2009, aiming to identify the possible geographic origins of isolates. Maximum-likelihood-based phylogenetic analyses of New Zealand and overseas TSWV isolates placed the L and M subgenome sequences of two isolates (MAF04 and PFR04) in distinct clades composed primarily of Korean, Japanese and Chinese isolates, in contrast to the remaining 21 isolates, which clustered with a cosmopolitan group of isolates. The nucleocapsid (N) gene sequences of MAF04 and PFR04 plus MAF02 clustered with Japanese isolates. Consequently, we postulate that these isolates may represent a distinct incursion into New Zealand, but we do not have enough evidence to indicate an incursion pathway. Alternately, these isolates may have arrived with an incursion that included a mixture of TSWV isolates of diverse international origins. The sequences of four of the TSWV isolates contained a number of sites with a mixture of nucleotides, suggesting that these isolates either consisted of several sequence variants or were from plants with mixed infections. One isolate (MAF02) was shown to be a either a reassortant or an S subgenome recombinant. Large amounts of low-level polymorphism were detected with low amino acid change fixation rates (purifying selection). Negative selection was indicated at four amino acid sites in the New Zealand TSWV N gene sequences.

Diversity and evolutionary history of lettuce necrotic yellows virus in Australia and New Zealand

Lettuce necrotic yellows virus (LNYV) is the type member of the genus Cytorhabdovirus, family Rhabdoviridae, and causes a severe disease of lettuce (Lactuca sativa L.). This virus has been described as endemic to Australia and New Zealand, with sporadic reports of a similar virus in Europe. Genetic variability studies of plant-infecting rhabdoviruses are scarce. We have extended a previous study on the variability of the LNYV nucleocapsid gene, comparing sequences from isolates sampled from both Australia and New Zealand, as well as analysing symptom expression on Nicotiana glutinosa. Phylogenetic and BEAST analyses confirm separation of LNYV isolates into two subgroups (I and II) and suggest that subgroup I is slightly older than subgroup II. No correlation was observed between isolate subgroup and disease symptoms on N. glutinosa. The origin of LNYV remains unclear; LNYV may have moved between native and weed hosts within Australia or New Zealand before infecting lettuce or may have appeared as a result of at least two incursions, with the first coinciding with the beginning of European agriculture in the region. The apparent extinction of subgroup I in Australia may have been due to less-efficient dispersal than that which has occurred for subgroup II – possibly a consequence of suboptimal interactions with plant and/or insect hosts. Introduction of subgroup II to New Zealand appears to be more recent. More-detailed epidemiological studies using molecular tools are needed to fully understand how LNYV interacts with its hosts and to determine where the virus originated.

The complete nucleotide sequence and genome organisation of a novel member of the family Betaflexiviridae from Actinidia chinensis

We report the complete genome sequence of a novel virus, tentatively named “actinidia seed-borne latent virus” (ASbLV), isolated from Actinidia chinensis in Auckland, New Zealand. The complete genome of ASbLV is 8,192 nucleotides long, excluding the 3ʹ poly(A) tail, contains four open reading frames, and is most closely related to Caucasus prunus virus (56% nucleotide sequence identity), a member of the genus Prunevirus. Based on the demarcation criteria of the family Betaflexiviridae, ASbLV is a new member of the genus Prunevirus.

Red clover vein mosaic virus—A Novel Virus to New Zealand that is Widespread in Legumes

Red clover vein mosaic virus (RCVMV) is an important virus of leguminous crops that can cause devastating losses. During a routine survey of legumes conducted on the South Island of New Zealand, RCVMV was found in mixed infections in clover plants with Alfalfa mosaic virus and White clover mosaic virus. The full-length sequence of the New Zealand isolate RCVMV-NZ from clover shared 96% nucleotide sequence identity with a chickpea isolate previously described from Washington (United States). Targeted surveys of pea, faba bean, and pasture crops showed that RCVMV-NZ is widespread on the South Island in New Zealand. This isolate is causing mild if any symptoms on experimental hosts and naturally infected plants.

Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australias grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

The truth about Pea mild mosaic virus

Pea mild mosaic virus (PMiMV) was reported from pea (Pisum sativum) in New Zealand in 1972. As this remains the only report of this virus worldwide, we aimed to try and clarify the status of PMiMV in New Zealand. In this study, nine species of comovirus were tested against antisera believed to have been raised to PMiMV and other comoviruses in 1972, and against current commercially available antisera. The ELISA results suggest that PMiMV is in fact Broad bean stain virus (BBSV). RT-PCR using novel generic comovirus primers confirmed the identity of each virus species to be true, and immunocapture RT-PCR using novel BBSV-specific primers confirmed that the BBSV isolate was not co-infected with another comovirus species. The generic primers proved to be useful in detecting all nine species of comoviruses commercially available, and in silico analysis of comovirus RNA-1 sequences suggested the primers should detect all comovirus species currently described.

First report of Tomato mosaic virus in Griselinia lucida, an epiphytic shrub native to New Zealand

Virus-like symptoms were observed on leaves of Griselinia lucida, a New Zealand native shrub, on Rangitoto Island, New Zealand. Rod-shaped particles similar to those of tobamoviruses were observed by electron microscopy and were identified by reverse transcription polymerase chain reaction as Tomato mosaic virus (ToMV). This is the first record of a virus in G. lucida and the first definite report of ToMV in New Zealand.

First Report of Wisteria vein mosaic virus on Wisteria sinensis in New Zealand

Wisteria vein mosaic virus (WVMV) is a member of the Potyvirus genus. The virus has been reported in Wisteria spp. in Australia, China, the United States, and a number of European countries (2). In 2006, several W. sinensis plants with mottling and mosaic symptoms were observed in a commercial plant nursery in Whenuapai, north of Auckland, New Zealand. These plants had been propagated from a nursery in the New Plymouth area of New Zealand. Sap from the symptomatic Wisteria plants was examined with an electron microscope and elongated and flexuous potyvirus-like particles approximately 750 nm long were observed. RNA was extracted from the symptomatic plants with a Qiagen RNeasy Plant Mini Kit (Doncaster, Australia). The RNA was initially tested using general potyvirus primers, PV1/SP6 (4) and U335 (3), with the cycling conditions of 94°C for 5 min followed by 40 cycles of 94°C for 45 s, 50°C for 45 s, 72°C for 90 s, and a final extension of 72°C for 7 min. The polymerase chain reaction (PCR) product (695 bp) was directly sequenced (GenBank Accession No. EU580146) and a BLAST search in GenBank showed 98% nucleotide identity with WVMV (GenBank Accession no. AF484549). The RNA was then tested using WVMV-specific primers, WVMVF1 and WVMVR1, and the published cycling conditions (2). PCR amplicons of 701 bp were obtained. PCR products were directly sequenced (GenBank Accession No. EU308592), and a BLAST search in GenBank showed 98% nucleotide identity with published sequences of WVMV (GenBank Accession Nos. AF484549 and AY656816). In addition, RNA was extracted from the original isolate of WVMV that was reported in the Netherlands (1; supplied by R. van der Vlugt, Plant Research International) and the RNA was amplified using the WVMV-specific primer pair. The sequence obtained from PCR amplicons of the type isolate (GenBank Accession No. EU308593) showed a 98% nucleotide identity with the New Zealand WVMV isolate and with published sequences of WVMV (as shown above). From the symptomatology, particle morphology, and nucleotide sequences, it is concluded that WVMV is present in New Zealand. The distribution of the virus in New Zealand is not known, but the affected plants at the New Plymouth nursery may have been imported into New Zealand as many as 30 years ago. Although WVMV infection can reduce the quality of commercial plants, the disease is not economically significant in New Zealand. References: (1) L. Bos. Neth. J. Plant Pathol. 76:8, 1970. (2) G. R. G. Clover et al. Plant Pathol. 52:92, 2003. (3) S. A. Langeveld et al. J. Gen Virol. 72:1531, 1991. (4) A. M. Mackenzie et al. Arch Virol. 143:903, 1998.