Preserving US microbe collections sparks future discoveries.

Collections of micro-organisms are a crucial element of life science research infrastructure but are vulnerable to loss and damage caused by natural or man-made disasters, the untimely death or retirement of personnel, or the loss of research funding. Preservation of biological collections has risen in priority due to a new appreciation for discoveries linked to preserved specimens, emerging hurdles to international collecting and decreased funding for new collecting. While many historic collections have been lost, several have been preserved, some with dramatic rescue stories. Rescued microbes have been used for discoveries in areas of health, biotechnology and basic life science. Suggestions for long-term planning for microbial stocks are listed, as well as inducements for long-term preservation.

Primer modification improves rapid and sensitive in vitro and field-deployable assays for detection of high plains virus variants.

A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods-real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)-for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5' terminus, raising the primers' melting temperature (Tm; ca. 10°C) to make them compatible with RT-HDA (required optimal Tm = 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.

Highly Sensitive End-Point PCR and SYBR Green qPCR Detection of Phymatotrichopsis omnivora, Causal Fungus of Cotton Root Rot.

Phymatotrichopsis omnivora, the causal pathogen of cotton root rot, is a devastating ascomycete that affects numerous important dicotyledonous plants grown in the southwestern United States and northern Mexico. P. omnivora is notoriously difficult to isolate from infected plants; therefore methods for accurate and sensitive detection directly from symptomatic and asymptomatic plant samples are needed for disease diagnostics and pathogen identification. Primers were designed for P. omnivora based on consensus sequences of the nuclear ribosomal internal transcribed spacer (ITS) region of geographically representative isolates. Primers were compared against published P. omnivora sequences and validated against DNA from P. omnivora isolates and infected plant samples. The primer combinations amplified products from a range of P. omnivora isolates representative of known ITS haplotypes using standard end-point polymerase chain reaction (PCR) methodology. The assays detected P. omnivora from infected root samples of cotton (Gossypium hirsutum) and alfalfa (Medicago sativa). Healthy plants and other relevant root pathogens did not produce PCR products with the P. omnivora-specific primers. Primer pair PO2F/PO2R was the most sensitive in end-point PCR assays and is recommended for use for pathogen identification from mycelial tissue and infected plant materials when quantitative PCR (qPCR) is not available. Primer pair PO3F/PO2R was highly sensitive (1 fg) when used in SYBR Green qPCR assays and is recommended for screening of plant materials potentially infected by P. omnivora or samples with suboptimal DNA quality. The described PCR-based detection methods will be useful for rapid and sensitive screening of infected plants in diagnostic laboratories, plant health inspections, and plant breeding programs.

Development of a rapid, sensitive, and field-deployable razor ex BioDetection system and quantitative PCR assay for detection of Phymatotrichopsis omnivora using multiple gene targets.

A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

Comparative assessment of 5' A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity.

Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5' A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5' flaps. Results indicate that even if the performance of some primers can be improved substantially by 5' flap addition, not all primers will be similarly improved. Optimal 5' flap sequences are dependent on the primer sequences, and alter the primer's T m value. The manipulation of this feature may enhance primer's efficiency to increase the PCR sensitivity and DNA yield.

First Report of Wheat streak mosaic virus on Wheat in New Zealand.

In August of 2005, seeds of wheat (Triticum aestivum) breeding line 6065.3 tested positive for Wheat streak mosaic virus (WSMV; genus Tritimovirus) by a WSMV-specific reverse transcription (RT)-PCR assay (2). The sequence of the 200-bp amplicon (GenBank Accession No. FJ434246) was 99% identical with WSMV isolates from Turkey and the United States (GenBank Accession Nos. AF454455 and AF057533) and 96 to 97% identical to isolates from Australia (GenBank Accession Nos. DQ888801 to DQ888805 and DQ462279), which belong to the subclade D (1). As a result, an extensive survey of three cereal experimental trials and 105 commercial wheat crops grown on the South Island of New Zealand was conducted during the 2005-2006 summer to determine the distribution of WSMV. Wherever possible, only symptomatic plants were collected. Symptoms on wheat leaf samples ranged from very mild mosaic to symptomless. In total, 591 leaf samples suspected to be symptomatic were tested for WSMV by a double-antibody sandwich (DAS)-ELISA (DSMZ, Braunschweig, Germany). Of the 591 symptomatic samples, 81 tested positive. ELISA results were confirmed by RT-PCR with novel forward (WSMV-F1; 5'-TTGAGGATTTGGAGGAAGGT-3') and reverse (WSMV-R1; 5'-GGATGTTGCCGAGTTGATTT-3') primers designed to amplify a 391-nt fragment encoding a region of the P3 and CI proteins. Total RNA was extracted from the 81 ELISA-positive leaf samples using the Plant RNeasy Kit (Qiagen Inc., Chatsworth, CA). The expected size fragment was amplified from each of the 81 ELISA-positive samples. The positive samples represent 30 of 56 wheat cultivars (54%) collected from 28 of 108 sites (26%) sampled in the growing regions from mid-Canterbury to North Otago. These results suggest that WSMV is widespread in New Zealand both geographically and within cultivars. WSMV is transmitted by the wheat curl mite (Aceria tosichella) (3), which had not been detected in New Zealand despite repeated and targeted surveys. WSMV is of great economic importance in some countries, where the disease has been reported to cause total yield loss (3). Although WSMV is transmitted by seeds at low rates (0.1 to 0.2%) (4), it is the most likely explanation of the spread of the disease in New Zealand. References: (1) G. I. Dwyer et al. Plant Dis. 91:164, 2007. (2) R. French and N. L. Robertson. J. Virol. Methods 49:93, 1994. (3) R. French and D. C. Stenger. Descriptions of Plant Viruses. Online publication. No. 393, 2002. (4) R. A. C. Jones et al. Plant Dis. 89:1048, 2005.

First Report of Spinach latent virus in Tomato in New Zealand.

A Lycopersicon esculentum (tomato) plant from a commercial property in New Zealand was submitted to the Investigation and Diagnostic Centre for diagnosis in 2003. Fruits had faint yellow ringspots but no obvious symptoms were observed on leaves. No virus particles were observed from tomato and symptomatic herbaceous plants crude sap preparations. Mechanically inoculated Nicotiana clevelandii and N glutinosa developed systemic chlorosis, whereas pinpoint necrotic local lesions were observed on Chenopodium amaranticolor. Chlorotic local lesions were also observed on C. quinoa followed by systemic necrosis. No symptoms were observed on Cucumis sativus, Gomphrena globosa, N. benthamiana, N. sylvestris, or N. tabacum cv. White Burley. Total RNA was extracted from N. glutinosa and C. quinoa leaf samples using the Qiagen (Qiagen Inc., Valencia, CA) Plant RNeasy Kit. Reverse transcription (RT) was carried out by using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen, Frederick, MD) followed with PCR using broad-detection primers targeting the genera Carmovirus, Dianthovirus, Ilarvirus, Tospovirus, (Agdia Inc., Elkhart, IN) and Tombusvirus (2). A positive RT-PCR amplification was obtained only with Ilarvirus primers. The 450-bp product (GenBank Accession No. DQ457000) from the replicase gene had a 97.4% nt and 98.6% aa identity with Spinach latent virus (SpLV; Accession No. NC_003808). An RT-PCR protocol was developed for the specific detection of SpLV. Primers were designed from three SpLV RNA sequences (RNA1: NC_003808; RNA2: NC_003809; RNA3: NC_003810) using the Primer3 software (3). Primers SpLV-RNA1-F (5'-TGTGGATTGGTGGTTGGA-3') and SpLV-RNA1-R (5'-CTTGCTTGAGGAGAGATGTTG-3') anneal to the replicase gene from nt 1720 to 2441. Primers SpLV-RNA2-F (5'-GAACCACCGAAACCGAAA-3') and SpLV-RNA2-R (5'-CCACCTCAACACCAGTCATAG-3') bind to the polymerase gene from nt 603 to 1038. Primers SpLV-RNA3-F (5'-GCCTTCATCTTTGCCTTTG-3') and SpLV-RNA3-R (5'-CATTTCATCTGCGGTGGT-3') amplify the movement protein gene from nt 724 to 936. The predicted amplified product sizes were 722, 436, and 213 bp from RNA1, RNA2, and RNA3, respectively. RT was carried out as described above. PCR was performed in a 20-µl reaction containing 2 µl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of forward and reverse primers, and 1 U Taq polymerase (Promega, Madison, WI). The PCR amplification cycle was identical for the three primer pairs: denaturation (95°C for 3 min) followed by 37 cycles of 95°C (20 s), 60°C (30 s), and 72°C (30 s) with a final elongation step (72°C for 3 min). The amplified products were analyzed by gel electrophoresis, stained with SYBR Green, and their identities confirmed by sequencing. The tomato sample was grown from seed imported from the Netherlands where SpLV occurs (4). The virus is of potential importance for the tomato industry because of its symptomless infection and high frequency of seed transmission in many plant species (1,4). SpLV has never been detected in other submitted tomato samples. Consequently, SpLV is not considered to be established in New Zealand. To our knowledge, this is the first report of SpLV in tomato. References: (1) L. Bos et al. Neth. J. Plant Pathol. 86:79, 1980. (2) R. Koeing et al. Arch. Virol. 149:1733, 2004. (3) S. Rozen and H. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols. Humana Press, Totowa, NJ, 2000. (4) Z. Stefenac and M. Wrischer. Acta Bot. Croat. 42:1, 1983.

Detection of Poinsettia mosaic virus by RT-PCR in Euphorbia spp. in New Zealand.

Euphorbia pulcherrima (poinsettias) are commonly infected with Poinsettia mosaic virus (PnMV), which resembles the Tymovirus genus in its morphology and viral properties (2) but is closer to the Marafivirus genus at the sequence level (1). Symptoms induced by PnMV range from leaf mottling and bract distortion to symptomless (2). The presence of PnMV in plants imported into New Zealand had never been proven. Leaves of 10 E. pulcherrima samples and six samples from other Euphorbia spp. (E. atropurpurea, E. lambii, E. leuconeura, E. mellifera, E. milii, and E. piscatorial) were collected in the Auckland area, North Island in 2002. Isometric particles of 26 to 30 nm in diameter were observed with electron microscopy in 3 of 10 E. pulcherrima samples. These three samples produced systemic chlorosis and crinkling symptoms on mechanically inoculated Nicotiana benthamiana, which tested PnMV positive by double-antibody sandwich (DAS)-ELISA (Agdia, Elkart, IN). No particles or symptoms on N. benthamiana were observed with the other Euphorbia spp., which were also PnMV-negative by DAS-ELISA. A reverse transcription-polymerase chain reaction (RT-PCR) was developed to further characterize PnMV. Specific primers were designed from the PnMV complete genome sequence (Genbank Accession No. AJ271595) using the Primer3 web-based software (4). Primer PnMV-F1 (5'-CCTGTATTGTCTCTTGCCGTCC-3') and primer PnMV-R1 (5'-AGAGGAAAGGAAAAGGTGGAGG-3') amplified a 764-bp product from nt 5291 of the 5'-end RNA polymerase gene to nt 6082 of the 3'-untranslated region (UTR). Total RNA was extracted from leaf samples using the Qiagen Plant RNeasy Kit (Qiagen Inc., Chastworth, CA). RT was carried out by using PnMV-R1 primer and MMLV reverse transcriptase (Promega, Madison, WI). The PCR was performed in a 20-µl volume reaction containing 2 µl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM PnMV-F1 primer, and 1 U of Taq polymerase (Promega) with a denaturation step (94°C for 5 min), 30 amplification cycles (94°C for 30 s; 55°C for 30 s; 72°C for 1 min), and a final elongation (72°C for 5 min). The sequence of the RT-PCR product (Genbank Accession No. DQ462438) had 98.7% amino acid identity to PnMV. PCR products were obtained from two of three PnMV ELISA-positive E. pulcherrima and three of three PnMV ELISA-positive symptomatic N. benthamiana. The failure to amplify the fragment from all ELISA-positive PnMV is likely because of the presence of inhibitors and latex in E. pulcherrima (3) that make the RNA extraction difficult. Thus, while RT-PCR may be useful for further characterizing PnMV isolate sequences, ELISA may be more reliable for virus detection. In conclusion, to our knowledge, this is the first report of PnMV in E. pulcherrima but not in other Euphorbia spp. in New Zealand. E. pulcherrima plants have been imported into New Zealand for nearly 40 years, and the virus is probably widespread throughout the country via retail nursery trading. References: (1) B. G. Bradel et al. Virology 271:289, 2000. (2) R. W. Fulton and J. L. Fulton. Phytopathology 70:321, 1980. (3) D.-E. Lesemann et al. Phytopathol. Z. 107:250, 1983. (4) S. Rozen and S. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols: Methods in Molecular Biology. S. Krawetz and S. Misener, eds. Humana Press, Totowa, NJ, 2000.

Partial Characterization of a Carla-Like Virus Infecting Yam (Dioscorea spp.) from China.

Dioscorea opposita (yam) from China was tested for viruses during post-entry quarantine in New Zealand during 2004. No obvious symptoms or virus particles were observed from yam. Mechanically inoculated Nicotiana occidentalis cvs. 37B and P1 produced systemic chlorosis, leaf reduction, and stunting, whereas no symptoms were observed on other tested herbaceous plants (Chenopodium amaranticolor, C. quinoa, Cucumis sativum, Gomphrena globosa, N. benthamiana, N. clevelandii, N. glutinosa, N. sylvestris, and N. tabacum cv. White Burley). Numerous filamentous particles (approximately 600 nm long) were observed by using electron microscopy from symptomatic N. occidentalis. Total RNA was extracted from yam and symptomatic N. occidentalis leaf samples using the Qiagen Plant RNeasy kit (Qiagen, Valencia, CA). Reverse transcription (RT) was carried out using random hexamer primers and SuperScript II RNase H¯ reverse transcriptase (Invitrogen, Carlsbad, CA) followed by polymerase chain reaction (PCR) with different primer pairs. Samples tested negative for Chinese yam necrotic mosaic virus (ChYNMV; genus Macluravirus) with specific primers (supplied by T. Kondo, Aomori Green BioCenter, Aomori, Japan). Negative results were also obtained for the genera Potyvirus, Potexvirus, Capillovirus, Trichovirus, and Foveavirus using RT-PCR with broad detection primers (1,2,4). A positive RT-PCR amplification was obtained from the yam and N. occidentalis samples with universal primers for the genus Carlavirus (Agdia Inc., Elkhart, IN). The 275-bp amplified products from the viral replicase were cloned and sequenced. The yam virus shows a high amino acid similarity with Hop latent virus (87.9%), Aconitum latent virus (86.8%) and Potato virus M (86.8%). Filamentous virus particles belonging to the genera Macluravirus, Potyvirus, and Potexvirus have been reported in yam (3). These virus species are not associated with the carlavirus infection since the virus found in D. opposita tested negative using RT-PCR with primers for these genera. There are no carlaviruses reported to be infecting yams, therefore, it may be considered as a new host-virus association. References: (1) X. Foissac et al. Acta Hortic. 550:37, 2001. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) B. S. M. Lebas. Ph.D. thesis. Greenwich University, Chatham Maritime, UK, 2002. (4) R. A. A. Van der vlugt and M. Berendsen. Eur. J. Plant Pathol. 108:367, 2002.

Development of an RT-PCR for High Plains virus Indexing Scheme in New Zealand Post-Entry Quarantine.

High Plains virus (HPV) causes a potentially serious economic disease of cereals and is of quarantine importance for New Zealand. HPV is transmitted by the wheat curl mite Aceria tosichella, and neither the virus nor its vector is present in New Zealand. Cereal seeds imported to New Zealand are required to be certified HPV-free, as the virus is a regulated pest. A procedure was developed for inspecting plants and testing cereal seedlings in quarantine using reverse transcriptase polymerase chain reaction (RT-PCR) as a detection method. A sample of 50,655 sweet corn seeds was taken from an imported commercial line and germinated in containment. Symptomatic seedlings were collected at 3 and 4 ½ weeks after sowing. Eight out of 27 symptomatic samples tested HPV positive by RT-PCR and were confirmed by enzyme-linked immunosorbent assay (ELISA). Sequence analysis revealed that the HPV isolates had a 99.3 to 100% nucleotide identity and 99.0 to 100% amino acid similarity with the HPV USA isolate (GenBank accession no. U60141). HPV variants were detected by single stranded conformational polymorphism (SSCP) analysis but not by restriction fragment length polymorphism (RFLP).