Summary
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.
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 Tₘvalue. The manipulation of this feature may enhance primer’s efficiency to increase the PCR sensitivity and DNA yield.
Tobacco mosaic virus (TMV), Hosta virus X (HVX), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are a few of the major viruses that infect ...ornamental and nursery plants. These viruses cause significant losses that impact growers and the ornamental industry. Often, a single ornamental plant is co‐infected by more than one virus, which makes identification and discrimination of these viruses a difficult task, thus creating delays and limiting regulatory measures for effective quarantine. The aim of this study is to develop a sensitive, rapid, economic, and reliable multiplex Reverse Transcription PCR (RT‐PCR) for simultaneous detection and discrimination of these five viruses. Specific PCR primers were designed using the consensus sequences of corresponding capsid protein (CP) genes of HVX and CMV, the nucleocapsid protein (NP) genes of TSWV and INSV, and the movement protein (MP) and CP genes of TMV. The primers were validated in vitro using single and multiplex RT‐PCR assays. The detection limit of each primer set in multiplex RT‐PCR was 100 fg (TMV), 1 fg (HVX), 10 fg (CMV), 10 pg (TSWV) and 10 pg (INSV). Forty‐six infected nursery samples collected from different locations in the USA were screened for virus infections using this multiplex RT‐PCR. The multiplex RT‐PCR has a potential for its application in routine diagnostics, quarantine, and epidemiological studies. The developed method is reliable, sensitive, and economic for testing a wide range of ornamental and nursery plants for detection of these viruses.
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.
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 1/2 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).
Soil-borne wheat mosaic virus (SBWMV) was identified using transmission electron microscopy, enzyme-linked immunosorbent assay (ELISA) with SBWMV-specific antibodies and reverse ...transcription-polymerase chain reaction (RT-PCR) tests with SBWMV-specific primers and sequence comparison in two out of five samples collected from a wheat ( Triticum aestivum ) plant showing severe leaf mosaic. The plant was also infected with Barley stripe mosaic virus and Barley yellow dwarf virus -MAV and -PAV. A further 28 out of 200 wheat samples tested positive for SBWMV using ELISA. A comparison of 11 New Zealand SBWMV isolates indicated that they were all identical and had 98% nucleotide sequence identity with SBWMV isolates from the United States, subgroup New York-Illinois. At the location of the SBWMV outbreak the vector, Polymyxa graminis , was detected by PCR and the identity was confirmed by sequencing. This is the first report of SBWMV 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.
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