G. R. G. Clover

A New ‘Candidatus Liberibacter’ Species Associated with Diseases of Solanaceous Crops

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with Candidatus Liberibacter species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named Candidatus Liberibacter solanacearum. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

'Candidatus Liberibacter solanacearum', associated with plants in the family Solanaceae

A liberibacter (isolate NZ082226) was detected in a symptomatic tomato plant and subsequently in five other members of the family Solanaceae: capsicum, potato, tamarillo, cape gooseberry and chilli. Phylogenetic analyses of the 16S rRNA gene sequence, the deduced amino acid sequence of the rplJ gene and a partial nucleotide sequence of the beta operon indicated that isolate NZ082226 represents a novel candidate species of Candidatus Liberibacter, for which the name Candidatus Liberibacter solanacearum is proposed.

A new 'Candidatus Liberibacter' species in Solanum betaceum (Tamarillo) and Physalis peruviana (Cape Gooseberry) in New Zealand.

A new Candidatus Liberibacter species was recently identified in tomato, capsicum, and potato in New Zealand. The tomato/potato psyllid, Bactericera cockerelli, is thought to be the vector of this species of liberibacter. During studies to determine additional host plants of the pathogen, leaves of Solanum betaceum (tamarillo, also known as tree tomato) and leaves and stems of Physalis peruviana (cape gooseberry) were collected from a home garden in South Auckland, New Zealand in July of 2008. These plants were not showing any obvious disease symptoms. They were located close to a commercial glasshouse site containing known liberibacter-infected tomatoes, and many psyllids were observed on the tamarillo tree over the summer and until late autumn. Total DNA was extracted from four tamarillo and two cape gooseberry samples with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples from tamarillo that were used for the extraction were taken from the midveins of old and young leaves and from young petioles. For cape gooseberry, samples were from the leaf midveins and the stems. The samples were tested by PCR using primers OA2 (GenBank Accession No. EU834130) and OI2c (1). These primers amplify a 1,160-bp fragment of the 16S rRNA gene of the new liberibacter species. Amplicons of the expected size were obtained from all four tamarillo samples, with no amplification from negative control tamarillo plants grown from seed in an insect-proof glasshouse. Almost the entire length of the 16S rRNA gene was amplified using primer pairs fD2 (3)/OI2c and OA2/rP1 (3), and the 16S-23S rRNA intergenic spacer was amplified with primer pair OI2/23S1 (2). These amplicons, along with that from the OA2/OI2c primer pair, were directly sequenced, and overlapping fragments were assembled using the SeqMan software of the LaserGene package (DNASTAR, Inc., Madison, WI) (GenBank Accession No. EU935004). A 650-bp fragment of the β operon was also amplified and sequenced directly (GenBank Accession No. EU935005). BLAST analysis showed 100% nt identity to the liberibacter of tomato (GenBank Accession Nos. EU834130 and EU834131) and potato (GenBank Accession Nos. EU849020 and EU919514). The two cape gooseberry samples produced amplicons of the expected size with the 16S rRNA and β operon primers and the origin of the fragments were confirmed by direct sequencing with BLAST analysis showing 100% nt identity to isolates from tomato, potato, and tamarillo. To determine the distribution of disease, 53 samples of 10 leaves each (representing two leaves from five plants) were collected randomly from a commercial tamarillo crop in South Auckland. Small sections of the midveins were removed from each of the 10 leaves, bulked, and DNA was extracted as described above. The samples were tested by PCR using primer pair OA2/OI2c. Amplicons of the expected size were obtained from 2 of the 53 samples. To our knowledge, this is the first report of a liberibacter in tamarillo and cape gooseberry. It is unknown if the liberibacter induces symptoms in these species or if they act as symptomless reservoirs of the pathogen. The infected plants will be observed for symptom development over the course of a growing season. References: (1) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 47:224, 1997. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

First Report of ‘Candidatus Phytoplasma australiense’ in Potato

In January of 2009, potato plants (Solanum tuberosum) from a commercial crop in the Waikato Region, New Zealand were observed to have symptoms of upward rolling and purpling of the leaves. The symptoms appeared similar to those of zebra chip, a disorder of potato recently found to be associated with Candidatus Liberibacter solanacearum in New Zealand and the United States (4). Total DNA from the leaf midveins and tubers from one of the symptomatic plants was separately extracted with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). DNA extracted from leaf midveins and tubers tested negative for Ca. L. solanacearum by nested-PCR using primer pair OA2/OI2c (4) followed by Lib16S01F/Lib16S01R (5-TTCTACGGGATAACGCACGG-3 and 5-CGTCAGTATCAGGCCAGTGAG-3), which amplifies a 580-bp region of the 16S rRNA gene. However, DNA extracted from the tuber tissue tested positive for phytoplasma by TaqMan real-time PCR (3). No phytoplasma was detected in the DNA extracted from leaf tissue. The 16S rRNA gene, 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene of the phytoplasma were amplified with primers P1/P7 (1). The PCR product was cloned into the pCR 4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. FJ943262). BLAST analysis showed 100% identity to Ca. Phytoplasma australiense (16SrXII, Stolbur group). A fragment of approximately 850-bp of the Tuf gene was also amplified (2) and sequenced directly (GenBank Accession No. FJ943263). BLAST analysis showed 100% identity to Tuf gene variant IX of Ca. P. australiense (2). An additional 14 plants showing similar leaf symptoms and also production of aerial tubers were collected from seven different potato fields from the Auckland and Waikato regions. Total DNA from the leaf midveins, stem, and tubers were separately extracted from each of the plants. The samples were tested for phytoplasma by nested-PCR using primer pair R16F2/R16R2, followed by NGF/NGR (1), and tested for Ca. L. solanacearum by nested-PCR as described above. Seven plants tested positive only for phytoplasma, three tested positive for only Ca. L. solanacearum, and four plants tested positive for both pathogens. The pathogens were most commonly detected in samples extracted from the stem with 9 and 5 of the 14 samples testing positive for phytoplasma and liberibacter, respectively. Six of each of the leaf and tuber samples tested positive for phytoplasma. Liberibacter was detected in one of the leaf samples and in four of the tuber samples. Ca. P. australiense has only been reported from New Zealand and Australia. The only other known hosts of Ca. P. australiense in New Zealand are strawberry and native plants belonging to the genera Cordyline, Coprosma, and Phormium (2). In Australia, Ca. P. australiense is associated with Australian grapevine yellows and Papaya dieback (2). To our knowledge, this is the first report of Ca. P. australiense infecting potato as well as the first report of phytoplasma and Ca. L. solanacearum mixed infections in potato. References: (1) M. T. Andersen et al. Plant Pathol. 47:188, 1998. (2) M. T. Andersen et al. Phytopathology 96:838, 2006. (3) N. M. Christensen et al. Mol. Plant Microbe Interact. 17:1175, 2004. (4) L. W. Liefting et al. Plant Dis. 93:208, 2009.

Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection.

A diagnostic test incorporating reverse-transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR) was developed for the detection of Beet necrotic yellow vein virus (BNYVV). The RT-PCR used the primers designed by (Henry et al., J. Virol. Methods 54 (1995)15) but refinements were made to the protocol including simplification of the extraction method, the use of standard reagents and adoption of a one-step procedure. None of these changes impaired sensitivity or specificity. The RT-PCR could also be used to amplify immunocaptured virus but this was slightly less sensitive than amplification from purified RNA. In nPCR, a second round of amplification was performed using primers, which produce a specific 326 base-pair product. Both RT-PCR and nPCR detected a range of 21 isolates collected from Europe, America and Asia (including A, B and P pathotypes) isolated from either sugar beet or Chenopodium quinoa. Neither assay produced PCR products using total RNA extracted from the roots of healthy sugar beet or beet infected with Beet soil-borne virus. However, the sensitivity of the nPCR was 1000 times greater than the standard RT-PCR. The reliability of the standard RT-PCR and nPCR was demonstrated using a range of cultivars collected from an infected field site. The use of the nPCR assay is recommended for applications where its improved sensitivity over standard RT-PCR is necessary, for example in the early detection of infection from bait-test soils and for quarantine and breeding purposes.

First report of Grapevine yellow speckle viroid 1 and Hop stunt viroid in grapevine (Vitis vinifera) in New Zealand.

In February 2009, grapevines (Vitis vinifera) in a commercial vineyard in Auckland were showing shortened, spindly canes with tiny leaves. Approximately 10% of the vines were affected. An RNeasy Plant Mini Kit (Qiagen, Valencia, CA) was used to isolate total RNA from leaves collected from six symptomatic (cvs. BAC0022A and Syrah) and eight symptomless vines (cvs. BAC0022A, Syrah, and Chardonnay). RNA was tested by reverse transcription-PCR for the presence of Australian grapevine viroid, Citrus exocortis viroid, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2, and Hop stunt viroid (HSVd). Four of the six symptomatic and all the symptomless vines tested positive for GYSVd-1 using primers 5-TGTGGTTCCTGTGGTTTCAC-3 and 5-ACCACAAGCAAGAAGATCCG-3, which amplify the complete genome (368 bp), and published primers PBCVd100C/194H (3), which amplify a 220-bp region of the genome. Amplicons from each PCR were transformed into a pCR 4-TOPO vector (Invitrogen, Carlsbad, CA), cloned, and sequenced. Sequence from both PCRs aligned identically to generate a consensus sequence (GenBank Accession No. HQ447056), which showed 99% nt identity to GYSVd-1 (GenBank No. X87906) by BLASTN analysis. All symptomatic and symptomless vines also tested positive for HSVd using primers C/H-HSVd (4) and HSVd-C60/H79 (1), which amplify the complete genome (298 bp). Amplicons from each isolate were cloned and sequenced. Sequence from both PCRs were aligned. Clones from all isolates, with the exception of one, aligned identically to create a consensus sequence (GenBank No. HQ447057) that showed 99% nt identity to Chinese HSVd isolates from grapevine (GenBank Nos. DQ371436-59) by BLASTN analysis. Sequence from the remaining isolate (GenBank No. HQ447056) was identical to a German Riesling grape isolate of HSVd (GenBank No. X06873). The presence of each viroid was further confirmed in PCR-positive plants by dot-blot hybridization with digoxigenin-labeled synthetic ssRNA probes specific to the full-length genomes of GYSVd-1 and HSVd (S. Harper and L. Ward, unpublished data). To our knowledge, this is the first report of GYSVd-1 and HSVd in V. vinifera in New Zealand. Since both viroids were detected in symptomatic and symptomless plants, the symptoms observed in the vineyard cannot be attributed to viroid infection. Symptoms described for GYSVd-1 include leaf spots and flecks, but no disease symptoms have been reported in grapes as a result of HSVd (2). Viruses found in the vines include Grapevine leaf roll virus-3, Grapevine viruses A and B, and Rupestris stem pitting associated virus, but these are not thought to be the cause of the symptoms. Two sequence types of HSVd were found, suggesting at least two separate introductions of HSVd into the vineyard. The vineyard is more than 40 years old so both viroids may have been present for some years. Export of wine from New Zealand was worth 1 billion dollars in 2009, so there is potential for these viroids to have an economic impact if symptoms are expressed. HSVd has been reported from China, Europe, Japan, Middle East, Pakistan, and the United States. GYSVd-1 has been reported from Australia, China, East Mediterranean, Europe, Japan, and the United States. References: (1) A. Hadidi et al. Acta Hortic. 309:339, 1992. (2) A. Hadidi et al., eds. Viroids. CSIRO Publishing, Collingwood, Australia, 2003. (3) R. Nakaune and M. Nakano. J. Virol. Methods 134:244, 2006. (4) A. M. Shamoul et al. J. Virol. Methods 105:115, 2002.

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.

New hosts of "Candidatus Phytoplasma australiense" in New Zealand

Abstract“Candidatus Phytoplasma australiense” occurs in New Zealand and Australia where it is associated with plant diseases in native, weed and crop plants. In New Zealand, this phytoplasma is historically associated with the diseases, Phormium yellow leaf, Strawberry lethal yellows, Cordyline sudden decline and Coprosma lethal decline. Between January 2009 and July 2010, four new hosts of “Ca. P. australiense” have been identified in New Zealand: potato (Solanum tuberosum), Jerusalem cherry (Solanum pseudocapsicum), swan plant (Gomphocarpus fruticosa) and celery (Apium graveolens), as well as a new disease association in boysenberry (Rubus hybrid). A 1.2xa0kb region of the 16S rRNA gene of the phytoplasma amplified from the new hosts was identical to each other. Partial tuf gene sequence analysis of 32 isolates from potato, Jerusalem cherry, swan plant, celery, boysenberry as well as from the Zeoliarus planthopper vector, revealed that they belonged to two separate subgroups, tuf variant VII and tuf variant IX. Two of the isolates, one from potato and the other from celery, contained a mixed infection of both phytoplasma subgroups.

First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) in New Zealand

In February 2009, 10 cape gooseberry plants (Physalis peruviana) grown from seed on a domestic property in Christchurch, New Zealand, showed severe leaf distortion, fasciation and etiolation of growing tips, and weak flowering. Symptoms were first observed in the emerging seedlings. No virus particles were observed in sap from infected plants with the electron microscope. Total RNA was isolated from leaves of the 10 plants with a Qiagen RNeasy Plant Mini Kit (Valencia, CA). All 10 plants tested positive for Potato spindle tuber viroid (PSTVd) by real-time reverse transcription (RT)-PCR (1) and by RT-PCR with PSTVd-specific primers (3) and generic pospiviroid primers (4). For both conventional PCRs, the expected 359-bp amplicons were sequenced directly and sequences were aligned together to create a consensus sequence (GenBank Accession No. FJ797614). BLASTn analysis showed 98% nucleotide identity to PSTVd (EU862231, DQ308556, X17268, and AY532801-AY532804). Sap from one of the infected plants was mechanically inoculated onto healthy P. peruviana, Solanum lycopersicum Rutgers, Chenopodium amaranticolor, C. quinoa, Cucumis sativum Crystal Apple, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis 37B, N. tabacum WB, N. sylvestris, and Phaseolus vulgaris Prince. After 4 weeks, the leaves of the Rutgers tomato plants were showing severe distortion, purpling, and necrosis of mid-veins and P. peruviana plants were showing distortion of newly emerging apical leaves. Healthy control P. peruviana were asymptomatic. Symptoms appeared milder than that observed in the original P. peruviana plants, but this may be related to different environmental conditions or age or growth stage of the plants when inoculated. All other indicator plants were symptomless, but along with P. peruviana, tested positive for PSTVd by real-time RT-PCR (1). The presence of PSTVd was further confirmed in one original symptomatic and the mechanically inoculated P. peruviana plants and in the indicator plants by dot-blot hybridization with a digoxygenin-labeled synthetic ssRNA probe specific to the full-length PSTVd genome. PSTVd has been reported in New Zealand previously in commercial glasshouse crops of tomatoes and peppers (2), but was eradicated and so remains a regulated pest. The plants were grown from seeds imported from Germany and it is possible that the infection was seedborne. PSTVd was reported in young cape gooseberry seedlings in Germany and Turkey but the infection was asymptomatic (5). Symptoms were associated with the PSTVd-infected cape gooseberry in New Zealand. To our knowledge, this is the first report of the viroid in domestically grown plants in New Zealand, and only the second report of PSTVd in cape gooseberry worldwide. Our findings suggest that this species is an emerging host for PSTVd and that dissemination of seed may provide a pathway for international movement of the viroid. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) B. S. M. Lebas et al. Australas. Plant Pathol. 34:129, 2005. (3) A. M. Shamoul et al. Can. J. Plant Pathol. 19:89, 1997. (4) J. T. H. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (5) J. T. H. Verhoeven et al. Plant Dis. 93:316, 2009.

Detection of European Isolates of Oat Mosaic Virus

Oat mosaic virus (OMV) is a fungally-transmitted virus which causes yield losses in winter oats in five European countries. Detection of the virus has depended upon the recognition of transient symptoms or electron microscopy. Recent research has confirmed that the virus is a Bymovirus, yet OMV could not be reliably detected by the enzyme-linked immunosorbent assay (ELISA) using a range of antisera raised against other members of the genus. Therefore, a reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed to specifically detect the virus. Using total RNA isolated from 16 field OMV isolates collected from throughout Europe, these primers were shown to reliably detect the virus in either one-step or two-step RT-PCR. The primers were specific and no PCR product was obtained with either Oat golden stripe virus (OGSV), which is frequently associated with OMV, or with other members of the Bymovirus genus. The two-step protocol was able to detect as little as 5 × 10−3μl (10ng) of total RNA isolated from an infected plant. Both protocols were as reliable as electron microscopy, but were more sensitive and were able to detect infection earlier than in mechanically-inoculated plants. However, this protocol did not detect three American isolates of the virus nor was amplification achieved using alternative primers raising the possibility that these isolates may represent a separate strain or virus. This protocol enables sensitive, rapid and reliable detection of OMV and will therefore assist management of the disease.