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Biodiversity of Antarctica includes climatically adapted microflora. Antarctic flora mainly contains cryptogams and are dominated by lichens, followed by mosses and algae. Among three bio-geographic zones of Antarctica, Sub-Antarctic zone is the most diverse and followed by Maritime and Continental zone. The Antarctic plant growth is largely limited to the coastal areas of Sub- and Maritime Antarctic regions. Two vascular plants; Deschampsia antarctica and Colobanthus quitensis are restricted along with other cryptogams at Antarctic Peninsula region. The average increase in temperature is the most prominent change in Antarctic Peninsula. Due to favorable conditions, cryptogams (lichens and mosses) are widely distributed in several localities of Antarctica. The algal growth is restricted to the semi permanent to permanent snow or melt ice and in floating water bodies of Antarctica. About 700 non-marine algal taxa were recorded from the different region of the Antarctica. Beside this, 200 additional taxa have been recorded from the continental Antarctica. The changes in temperature, pattern of cyclonic activity, availability of nutrients, water, high UV-B radiation along with other stresses posses obstacles for survival and distribution of Antarctic flora.

Surveys of commercial and seed potato fields for virus diseases (1998 to 2002) in Manitoba established that Potato virus Y (PVY) is of concern in seed potato production. To determine the prevalence of PVY strains, PVY-infected tubers identified by reverse transcription-polymerase chain reaction (RT-PCR) from surveys (2000 to 2001) were grown for symptom expression and strain characterization by strain-specific RT-PCR, bioassays, and serological assays. Of the samples collected (2000 to 2001) and tested by RT-PCR, 4.0% contained PVY. Further analysis of the PVY-positive samples by a duplex RT-PCR facilitating the simultaneous detection of common (PVYO) and tobacco veinal necrosis strains (PVYN/NTN) indicated that 37.5% contained PVYO and 63.5% contained PVYN-type isolates. Analysis of the PVYN-type samples using three monoclonal antibodies (MAbs) showed that all reacted with only the PVYO MAbs and not with the PVYN-specific MAb. Partial nucleotide sequences of both ends of PVY-RNA showed that the PVYN-type isolates resembled those reported in 1996 from Manitoba. These isolates are designated as PVYN:O. In view of the increased incidence of PVYN:O in one production area, seed tubers imported from other provinces of Canada and the neighboring United States were analyzed for PVYN:O. The PVYN:O was detected in imported seeds from Minnesota, Montana, and North Dakota.

Tomato chlorotic dwarf viroid (TCDVd), a member of the genus Pospivroid, family Pospiviroidae, was first identified on greenhouse tomato (Solanum lycopersicum) in Canada (2). Since then, it has also been reported elsewhere, e.g., on tomato in Colorado (4). During 2006 in Arizona, tomato plants in a large greenhouse facility with continuous tomato production exhibited viroid-like symptoms of plant stunting and chlorosis of the young leaves. Symptomatic plants were often located along the edge of the row, indicating the presence of a mechanical transmissible agent. Approximately 4% of the plants in this greenhouse were symptomatic in 2008. Symptoms were distinctly different from those caused by Pepino mosaic virus (PepMV), a virus that was generally present in this greenhouse and also in our test samples. Other commonly occurring tomato viruses were ruled out by serological, PCR, or reverse transcription (RT)-PCR tests in multiple laboratories. RT-PCR with two sets of universal pospiviroid primers, PospiI-FW/RE and Vid-FW/RE (4), yielded amplicons of the expected sizes of 196 and 360 bp in three samples collected from symptomatic plants. Direct sequencing of the amplicons revealed that the genome was 360 nt and 100% identical to the type TCDVd from Canada (GenBank Accession No. AF162131) (2). Mechanical inoculation with leaf tissue extract from four samples to plants of the tomato ‘Money-Maker’ resulted in the same viroid-like symptoms and TCDVd was confirmed in these plants by RT-PCR and sequencing. In both 2007 and 2008, 18 samples were tested using primers PSTVd-F and PSTVd-R (1), which are capable of amplifying the full TCDVd genome. Analysis of the sequences from the amplicons revealed two genotypes of TCDVd. The first genotype (GenBank Accession No. FJ822877) was identical to the type TCDVd and found in 11 samples from 2007 and one from 2008. The second genotype (GenBank Accession No. FJ822878) was 361 nt, differing from the first by nine nucleotide substitutions, 2 insertions, and 1 deletion. This second genotype was found in 7 and 17 samples from 2007 and 2008, respectively, and showed the highest sequence identity (97%) to a Japanese tomato isolate (AB329668) and a much lower sequence identity (92%) to a U.S. isolate previously identified in Colorado (AY372399) (4). The origin of TCDVd in this outbreak is not clear. The genotype identified first could have been introduced from a neighboring greenhouse where the disease was observed before 2006 and where this genotype also was identified in 2007. The second genotype may have been introduced from infected seed since TCDVd has recently been shown to be seed transmitted in tomato (3). To our knowledge, this is the first report of natural occurrence of TCDVd in Arizona. References: (1) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (2) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. (3) R. P. Singh and A. D. Dilworth. Eur. J. Plant Pathol. 123:111, 2009. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

A field isolate of Potato virus Y (PVY) was collected in New Brunswick, Canada in 2007 due to unusual symptoms observed on different potato cultivars. To unveil the PVY strain identity, tobacco and potato bioassays, PVY

North American potato production differs from other geographical regions such as Europe in that it is essentially a closed system, i.e., seed potatoes are not imported and production is dominated by only a few cultivars. The lack of significant seed imports provides a mechanism for seed certification to be extremely effective at minimizing virus levels in seed lots, especially if the changes in seed laws, postharvest testing, and tolerance limits discussed above are adopted. This is an opportunity to effectively manage PVY at levels that are at or below detection and well below economic significance. Aiding the seed certification programs in the adoption of the Canada/US-Management Plan for Potato Viruses that Cause Tuber Necrosis has and continues to build consensus and cooperation within the industry to reform and modernize seed certification practices and, as importantly, modernize best management practices that growers can implement so that their production meets or exceeds virus tolerances set within the seed certification standards. Seed inspectors could also benefit from continually updated information from the research community to help them better recognize the spectrum of symptoms caused by the various strains and variants of PVY in all the different cultivars now being grown in their states and provinces. They could also benefit from improved field diagnostics that will assist them and the growers in identifying problem plants that should be rogued. If PVY levels in seed can be minimized and on-farm management strategies can be optimized, then PVY incidence in the potato crop will be marginalized. The restricted distribution of the tuber necrotic strains also offers an opportunity to prevent these strains from becoming economically significant if appropriate testing of seed lots in those areas could prevent them from being planted. Shipping point inspections of tubers will also help in identifying and eliminating tuber necrotic viruses. The dominance of a few cultivars has been eroding in recent years. Russet Burbank, a cultivar introduced over 100 years ago, still accounts for 40 to 50% of the U.S. acreage, but acreage in the Northwestern United States has been declining steadily as other russet cultivars come on the market and gain acceptance. Potato cultivar has had a significant impact on the PVY problem, as with the release and widespread acceptance of Shepody, Russet Norkotah, and other asymptomatic carriers of PVY (http://oregonstate. edu/potatoes/latenttoPVYlist.htm), which in 2008 comprised more than 15 and 12% of the total U.S. and Canadian seed acreage, respectively. These cultivars have certainly contributed to the overall increase in PVY in the seed potato crop and by extension the commercial potato crop. The increased diversity of potato cultivars grown in both countries has also introduced a wider spectrum of PVY symptoms, most notably the milder symptoms that are characteristic of the PVYN/NTN and PVYN-Wi strains on many cultivars. Since the success of seed certification is dependent upon visual assessment of the crop, mild or absent symptoms means that many more infected plants go unnoticed. The more symptomatic PVYO strains are observed and removed, but the other strains remain in the crop and are passed along in the seed, contributing to an overall increase in PVY incidence and more importantly to a shift in PVY strain composition. The U.S. and Canadian potato industry stakeholders are increasingly aware of the PVY-associated challenges and have been moving rapidly to work with researchers and all aspects of the industry to implement plans to suppress PVY incidence. Continued education of growers, seed certification officials, and researchers alike, coupled with the development and adoption of new or revised best management practices and diagnostic tools, and the renewed inter est of breeders to develop virus resistant cultivars, will be the keys to success in bringing PVY incidence under control and in minimizing tuber necrotic strains.

Herein, we report an artificial nanoenzyme: CoSe2 with unique flake like morphology, exhibiting an excellent intrinsic peroxidase like activity. In comparison with naturally found Horseradish peroxidase (HRP), silver and gold based biomimetics, these nanoflakes mimics enhanced peroxidase like activity with higher Kcat and Vmax values for oxidation of both 3,3,5,5-tetramethylbenzidine (Kcat = 42.8 s−1, Vmax = 0.5139 μM/s) and o-phenylenediamine (Kcat = 84.95 s−1, Vmax = 1.0139 μM/s) under optimized pH conditions. A single electron transfer mechanism from TMB/OPD substrate favors formation of blue color TMB+/OX-OPD products. The unique flakes like morphology of CoSe2 nanoflakes provides active channels which offers better accessibility for the reactant, resulting in enhanced catalytic efficiency of CoSe2 nanoflakes. Additionally, there is no significant loss in the catalytic efficiency of CoSe2 nanoflakes even after 365 days which endorses higher thermal and chemical stability of these synthesized CoSe2 nanoflakes.

The effect of carrageenans on tomato chlorotic dwarf viroid (TCDVd) replication and symptom expression was studied. Three-week-old tomato plants were spray-treated with iota(ɩ)-, lambda(λ)-, and kappa(κ)-carrageenan at 1 g·L-1 and inoculated with TCDVd after 48 h. The λ-carrageenan significantly suppressed viroid symptom expression after eight weeks of inoculation, only 28% plants showed distinctive bunchy-top symptoms as compared to the 82% in the control group. Viroid concentration was reduced in the infected shoot cuttings incubated in λ-carrageenan amended growth medium. Proteome analysis revealed that 16 tomato proteins were differentially expressed in the λ-carrageenan treated plants. Jasmonic acid related genes, allene oxide synthase (AOS) and lipoxygenase (LOX), were up-regulated in λ-carrageenan treatment during viroid infection. Taken together, our results suggest that λ-carrageenan induced tomato defense against TCDVd, which was partly jasmonic acid (JA) dependent, and that it could be explored in plant protection against viroid infection.

Viroids are highly structured small circular RNAs that do not encode proteins but are able to replicate and cause diseases in plants. Characteristic symptoms caused by a viroid in a specific indicator host are the foundation of biological assay (bioassay) for detection and identification of the viroid. For bioassay of Solanaceae -infecting pospiviroids, a panel of indicator hosts are needed as similar types of symptoms may be induced by different viroids. For bioassay of citrus viroids, both citrus and herbaceous species are used. Many factors including environmental conditions, viroid strains, host species and genotype, and whether or not another viroid/virus is present, affect the bioassay. Combining bioassays with molecular assays for viroid detection and identification are also discussed.

A TaqMan real-time reverse transcription-PCR (real-time RT-PCR) procedure was developed, optimized, and compared with other routine methods to detect Potato virus Y (PVY) in dormant potato tubers. Three pairs of primers and probes were designed and evaluated for their suitability to facilitate the real-time RT-PCR detection of PVY for all strain groups including PVYO, PVYN, PVYN:O (= PVYN-Wi) and PVYNTN. Among the primer and probe combinations tested, the combination PVY-1 produced the lowest threshold cycle (Ct) value of 25.75. The procedure was further optimized by adjusting various parameters including primer/probe concentration, reaction volume, amplification cycles, and master mixes from different sources. The real-time RT-PCR was then employed to detect PVY from dormant tubers of different cultivars and potato fields, and the results were compared with those obtained from conventional RT-PCR, enzyme-linked immunosorbent assay (ELISA) on sprouts and grow-out testing. Out of 1,069 single-virus infected (PVY only) tubers tested, both formats of RT-PCR detected PVY in 52 samples, ELISA on sprouts in 45, ELISA on leaves in 54 and visual observations in 53. However, in 61 multiple-virus infected tubers tested, both formats of RT-PCR, and ELISA on both sprouts and leaves detected a similar number of positives, thus, making all the methods equally sensitive. Considering that ELISA requires sprouting of dormant potato tubers for PVY testing, grow-out testing takes approximately 6–8 weeks to obtain results, and conventional RT-PCR needs post-PCR processing, real-time RT-PCR offers a speedy alternative for large scale detection of PVY from dormant tubers. The method is therefore recommended for testing of PVY in potato tubers on a commercial scale in a diagnostic laboratory.

Laboratory assays demonstrated that wood ash is toxic to adult and larval stages of the Colorado potato beetle. Insect mortality reached 100% among larvae and beetles continuously exposed to wood ash for periods of up to 10 days but remained below 22% after 4 repeated topical applications. The application of ash reduced feeding and activity level by adult stages, at least initially. The significant control observed in the laboratory did not extend to field application. Observations suggest that wood ash is inactivated by moisture and that hydrophobic formulations of wood ash would be required for outdoor application as an insecticide. Adult beetles did not avoid crossing thin strips of ash in the laboratory, but preliminary observations suggest that thick layers of ash could act as a physical barrier (fence) limiting colonization by beetles.

We studied the mechanism of Ascophyllum nodosum (a brown macroalga) induced resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000. Root treatment of A. thaliana Col-0 plants with extracts of A. nodosum [aqueous (ANE), chloroform (C-ANE) and ethylacetate fractions, (E-ANE)] reduced the development of disease symptoms on the leaves. These extracts also induced resistance in salicylic acid deficient NahG and ics1 plants. However, the extracts did not elicit an effect on jar1 (jasmonic acid resistance 1) mutant. A. nodosum extract induced resistance to Pst DC3000 correlated with increased expression of jasmonic acid related gene transcripts PDF1.2 while PR1 and ICS1 expression were less affected. Additionally, pretreatment of Arabidopsis plants with ANE, protected the plants from a necrotroph, Sclerotinia sclerotiorum. The results suggest that the A. nodosum extracts can induce resistance in Arabidopsis to different pathogens which is largely jasmonic acid dependent.

A field isolate of Potato virus Y (PVY) was collected in New Brunswick, Canada in 2007 due to unusual symptoms observed on different potato cultivars. To unveil the PVY strain identity, tobacco and potato bioassays, PVYO and PVYN-specific antibody-based enzyme-linked immunosorbent assays, and reverse-transcription polymerase chain reaction (PCR)-based genotyping were carried out. All the assays demonstrated that the isolate, designated as PVYO-FL in this study, belonged to the PVYO strain group. Greenhouse tests with the potato cvs. FL 1533 and Jemseg confirmed the severe nature of infection by PVYO-FL. The complete genome sequences of PVYO-FL and PVYO-RB, the latter a mild PVYO isolate, were determined. BLAST analysis revealed that the two isolates shared 97 and 98% sequence identities at the nucleotide and polyprotein levels, respectively. Further BLAST analysis unveiled that PVYO-FL shared 99.7% nucleotide sequence identity with PVYO-Oz, an isolate reported in New York, United States, whereas the PVYO-RB isolate shared 99.2% sequence identity with PVYO-139, a PVYO isolate reported in New Brunswick, Canada. A phylogenetic tree of available, full-length sequences of PVY isolates demonstrated two subgroups within the PVYO branch, one clustered with PVYO-RB and the other with PVYO-FL. Group-specific sense primers for differentiation of the two subgroups were developed and evaluated. A limited survey of potato tubers collected from a field plot at the Potato Research Centre, Agriculture and Agri-Food Canada, using the newly developed PCR primers, indicated that 65.3 and 2.4% of the PVYO-positive tubers were infected with PVYO isolates belonging to the PVYO-FL and PVYO-RB subgroups, respectively. Assessment of the pathogenicity of three representative isolates from each subgroup on the potato cv. Jemseg demonstrated that severe and mild symptoms were induced by the PVYO-FL-like and PVYO-RB-like isolates, respectively.

The terrestrial ecosystem of Antarctica are among the most extreme on earth, challenging the communities and making their existence difficult by rapidly increasing annual summer influx of solar ultraviolet radiations (UV-R), extremely cold conditions and lesser availability of nutrients. Spring time ozone depletion is due to release of chlorofluorocarbons in the earth atmosphere and is a serious cause of concern among environmentalists. Antarctic continent is mostly dominated by cryptogamic plants with limited distribution in different parts of the icy continent however; their distribution is mostly confined to Sub-Antarctic region. By the virtue of light requirement, cryptogams are exposed to extreme seasonal fluctuation in photosynthetically active radiation (PAR), and ultraviolet (UV) radiation which are closely associated with photosynthetic pigments in photoautotrophic organisms. Antarctic cryptogams cope up the stress imposed by UV radiation by the development of efficient systems for repairing damage by synthesis of screening compounds such as UV-B absorbing pigments and anthocyanin compounds. A major part of the UV absorbing compounds are appeared to be constitutive in lichens which are usnic acid, perlatolic acid and fumarphotocetraric acid which is particularly induced by UV-B. Secondary metabolites such as phenolics, atranorin, parietin and melanin also enhance the plant defense, by different molecular targets in specific solar irradiance and potential for increased antioxidative protection to UV induced vulnerability.

Tomato chlorotic dwarf viroid (TCDVd) manually inoculated to transgenic (cv.‘Desiree’) potato plants containing antimicrobial cationic peptides failed to develop symptoms in above ground plant parts, but infected tubers were symptomatic. Plants from the infected tubers (second generation plants) emerged as either severely stunted (bushy stunt isolate, BSI) or tall and symptomless. Molecular characterization of BSI isolates showed TCDVd sequence variants 95 to 98% identical to TCDVd sequences from the database, while a viroid variant identical to TCDVd type isolate (acc # AF162131) was cloned from symptomless plants. The TCDVd BSI variants had novel U165C, GU177-178AA, and UCAC181-184CUUU nucleotide substitutions in the terminal right (TR) domain of the viroid molecule. The cloned viroid cDNAs of the BSI were infectious to experimental (cv. ‘Sheyenne’) tomato plants causing stunted plants with profuse auxiliary shoots. Visual evaluation of the susceptibility of the BSI to 18 potato and 21 tomato cultivars revealed severe symptoms in most cultivars of both species. The progeny variants accumulating in each potato and tomato cultivar exhibited the same novel TR domain in most cultivars, with only a slight variation in a few. The severity of the stunting symptoms induced by TCDVd from BSI isolates in both potato and tomato cultivars has not been noted previously with other TCDVd isolates and, as such, it is proposed that this new isolate be recognized as a distinct genotype. Emergence of this type of sequence variant in commercial fields or commercial tomato greenhouses could potentially cause relevant losses in both crops.

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.

Two novel aspects of Tomato chlorotic dwarf viroid (TCDVd) are reported, namely that TCDVd was detected in symptomless plants of Vinca minor, a trailing ground cover surviving at subzero temperatures (−12°C); and that TCDVd was seed-borne in tomato and detected in high percentages in tomato seeds and seedlings. Soaking seeds in a low concentration of sodium hypochlorite did not eliminate the viroid. The sequence analysis showed that the TCDVd isolate consists of 360 nucleotides and has sequence identity between 96% to 99% with isolates of TCDVd from other hosts.

Graft inoculation of potato (Solanum tuberosum) ‘Shepody’ plants with Potato virus A (PVA) results in hypersensitive resistance responses (HR) in tubers with characteristic necrosis. Increasing the number of PVA-containing scions or increasing the duration of scions on the ‘Shepody’ rootstock plants resulted in an increased number of tubers showing necrosis. Transgenic ‘Shepody’ plants with a sense or an antisense copy of PVA coat protein (CP) gene under the direction of the CaMV 35S promoter were developed. The plants were subjected to graft inoculation with PVA in the greenhouse and were monitored by detecting PVA using enzyme-linked immunosorbent assays and reverse transcriptase polymerase chain reaction during the growing season. No PVA was detected in the wild type or the antisense-CP transgenic lines. However, PVA was detected from one of the two sense-CP transgenic lines. Potato tubers from graft-inoculated plants were harvested 3 months after inoculation and were examined for necrosis. In...

Application of a simplified protocol for nucleic acid preparation for polymerase chain reaction (PCR) detection of greening bacterium (Candidatus Liberibacter asiaticus; Cla) and Citrus mosaic virus (CMBV) associated with citrus is described. Crude extracts of citrus tissues in NaOH - ethylenediaminetetraacetic acid solution, prepared without the use of liquid nitrogen, were spotted on a nitrocellulose membrane (NCM) and then eluted in water at 80 °C for 10 min. Both the greening bacterium and CMBV were detected by PCR from the eluted liquid from the spotted NCMs. The detection efficacy for the templates obtained in the NCM-eluted liquid was comparable with that of templates obtained with a multistep laboratory method or a commercial kit for nucleic acid preparation. The protocol is simple, inexpensive, rapid, and applicable to large-scale surveys of citrus trees. The spotted-membrane methodology can also be used for short-term sample storage for future testing or for long-distance sample transport to a d...

SummaryNesting of Apis dorsata Fabr. was influenced by the availability of forage plants and prevailing temperatures. There was an assured nectar and pollen supply during October to June through the flowering of more than twenty nectariferous plants, major ones being: Brassica campestris;Brassica rapa; Cajanus cajan; Moringa oleifera; Bombax ceiba; Helianthus annuus; Syzygium cumini; Emblica officinalis and Pongamia pinnata. The total numbers of nesting colonies were 28 in November and four in June coinciding with abundant food and optimum temperature in the first half and a decline later on. The greatest number of nests (35) was observed in January, at semi protected (32) and unprotected (3) sites, when minimum and maximum average temperatures were 7.8 and 24.2°C, respectively. The numbers of bee colonies at such sites in June, when the minimum and maximum temperatures were 26.8 and 33.7°C declined to four and none, respectively. May and June maximum temperature ranged between 40 to 45°C during daylight ...

Potato spindle tuber viroid (PSTVd) is a seed-transmissible viroid that can be present in floral parts of the host. However, a recent study (Y. Zhu, L. Green, Y.-M. Woo, R. Owens, and B. Ding. 2001. Virology, 279: 69–77), using Agrobacterium -mediated inoculation and in situ hybridization, has shown that PSTVd is present in sepals, but absent in petals, stamens, and ovary of infected tomato plants. In earlier studies, the presence of PSTVd in floral parts was determined by polyacrylamide gel electrophoresis and infectivity assays. In the present study, the presence of PSTVd and other species of the genus Pospiviroid in floral parts of tomato and other host species was studied using the highly sensitive reverse transcription – polymerase chain reaction. The reassessment showed that PSTVd was present in all floral parts of tomato, as evidenced by strong bands of polymerase chain reaction products. Moreover, floral-organ extracts containing the infectious viroid caused characteristic PSTVd symptoms in tomato...

Formats of a simple protocol for the preparation of nucleic acids for infectivity and RT-PCR detection of viroids from minute amounts of plant material are described. The method consists of preparing crude extracts in a NaOH-EDTA solution and then testing the supernatant. The NaOH-EDTA extract can be used at four distinct stages of preparation depending upon the accuracy desired, namely: (1) incubation of extract for 15 min at room temperature and the use of the supernatant for RT-PCR; (2) the supernatant can be spotted onto a nitrocellulose membrane (NCM) without vacuum, and the water-eluted liquid is used for RT-PCR; (3) centrifugation of the extract and use of supernatant in RT-PCR; (4) for quantitative accuracy, spotting the centrifuged supernatant on NCM using a vacuum device and then using the water-eluted liquid for RT-PCR. The protocols are rapid, inexpensive and applicable to large-scale epidemiological survey of ornamental plants or crops. The membranes are easily transported long distances and can be stored at room temperature for several months while retaining the ability to detect viroids by RT-PCR and by infectivity assays.

O Potato virus Y (PVY) tornou-se o maior problema nas áreas de produção de batata semente do Brasil. Somente as estirpes comum (PVYº) e necrótica (PVY N) eram detectadas infectando batata no Brasil. Esta situação mudou drasticamente a partir de 1997 quando um surto epidêmico de uma variante da estirpe necrótica de PVY causando arcos e anéis necróticos na superfície do tubérculo (PVY NTN) foi observado no país. Este estudo visou avaliar e validar uma metodologia de diferenciação de estirpes que causam necrose em tubérculos, proposta por Weilguny e Singh (1998). Vinte e oito isolados de PVY originários de tubérculos infectados de batata, provenientes de quatro estados brasileiros, foram analisados em sua reação em plantas de fumo, por Elisa, utilizando anti-soro policlonal e pelo método 3-primer RT-PCR. Quatro isolados induziram clareamento de nervuras e manchas peroladas em folhas de Nicotiana tabacum conforme esperado para a estirpe comum. Os 24 isolados restantes induziram necrose de nervuras neste hospedeiro portanto, classificados como da estirpe necrótica. Todos os 28 isolados de PVY reagiram positivamente contra o anti-soro policlonal de PVY por Elisa. Três métodos de extração de RNA foram testados, sendo que o método de hidrocloreto de guanidina mostrou-se o mais eficiente e de menor custo. Dos 28 isolados submetidos ao RT-PCR, um isolado de Santa Catarina e três do Rio Grande do Sul foram diferenciados como PVYº, confirmando os resultados do teste biológico. Um isolado de PVY N foi detectado no Estado de Santa Catarina e quatro no Rio Grande do Sul. O PVY NTN foi detectado em Minas Gerais (seis isolados), Santa Catarina (três isolados) e São Paulo (dez isolados). Estes resultados confirmam a presença dessa variante necrótica, PVY NTN, nas principais regiões produtoras de batata do Brasil.The Potato virus Y (PVY) has become the major virus problem in seed potato growing areas of Brazil. Only necrotic and ordinary PVY strains were found infecting potatoes in Brazil. This situation drastically changed around 1997 when an epidemic of a PVY necrotic variant causing necrotic rings on the potato tuber surface was observed in the country. This study aimed to test the tuber necrotic strain differentiation method proposed by Weilguny & Singh (1998). Twenty eight PVY isolates originated from infected tubers and leaves from four Brazilian States were analyzed by host reaction after inoculation in tobacco, by ELISA using polyclonal antiserum and by the 3-primer RT-PCR method. The ordinary strain type isolates induced vein clearing and chlorotic pearl spots on Nicotiana tabacum leaves. All 24 remaining isolates inducing vein necrosis in leaves were classified as necrotic strains. All 28 PVY isolates positively reacted to PVY polyclonal antiserum by Elisa. Three RNA extraction methods were compared and the guanidine hydrochloride method was the most efficient and of the lowest cost. One isolate from Santa Catarina State and three from Rio Grande do Sul out of 28 PVY isolates submitted to RT-PCR method were differentiated as PVYº, confirming the biological test. One isolate of PVY N was detected from Santa Catarina and four from Rio Grande do Sul State. The PVY NTN was detected in Minas Gerais (six isolates), Santa Catarina (three isolates) and São Paulo (ten isolates). These results confirmed the presence of this new variant, PVY NTN in the main potato growing areas of Brazil.

Nucleotide sequence analysis of polymerase chain reaction (PCR)-amplified pospiviroid-related fragments in several symptomless ornamental plants indicated the presence of three distinct viroid species: Iresine viroid (IrVd), Citrus exocortis viroid (CEVd), and Chrysanthemum stunt viroid (CSVd). Primers specific to viroid species, based on partial nucleotide sequences of the viroids, were used to clone full-length genomes of the three viroids from their original host species. Viroid isolates were cloned and sequenced from the following plants: Vinca major (IrVd and CSVd), a trailing Verbena sp. (IrVd and CEVd), and a double Impatiens sp. (two CEVd). The genome sizes of these viroids ranged from 355 to 375 nucleotides. For all three viroids, rod-like secondary structures with slight variations in the CSVd and IrVd isolates from Vinca can be predicted. Nucleotide substitution at positions 163 and 211 of IrVd indicated that U163 and A211 were critical in maintaining the perfect rod-like conformation. Pospivir...

Previous studies have identified a recombinant isolate group of the Potato virus Y (PVY), PVYN:O, which exhibits a PVYO serotype and a PVYN pathotype. Molecular fingerprinting indicated that the isolates possess a European (Eu)–PVYN/NTN-like P1 gene and one PVYN-PVYO recombinant joint at the HC/Pro-P3 site. In this study, the complete genomic RNA sequence of two isolates of PVYN:O was determined. The overall identities at the polyprotein level between PVYN:O and other isolates ranged from 94.1% to 97.2%. However, the identities varied from 70.9% to 100% when individual mature proteins were compared. Scanning the nucleotide sequences revealed that PVYN:O comprises a recombinant genome in which the segment of nucleotides 1 to ~2400 is from Eu-PVYN, and the remainder from PVYO. Analysis of a population of PVYN:O isolates indicated the existence of pathological variants that induced different symptoms level in tobacco plants: severe, 16.7%; intermediate, 66.7%; and mild, 16.7%. Three representative isolates w...

A rapid and simple protocol for preparing viral RNA from aphids ( Myzus persicae ) and potato ( Solanum tuberosum L.) tissue (leaves, sprouts, and tubers) for reverse transcription–polymerase chain reaction (RT–PCR) was developed. The four-step method involves: (1) preparing plant crude sap or aphid macerates in a buffered detergent (Triton XL-80N) solution; (2) immobilizing clarified sap on a nitrocellulose membrane; (3) performing reverse transcription using eluted water extract from cut-out spot from membrane; and (4) amplifying cDNA through PCR. The entire procedure from tissue grinding to RT–PCR can be completed within 6 h. The protocol was applied successfully for the detection of individual potato viruses: carlavirus Potato virus S (PVS), potexvirus Potato virus X (PVX), potyvirus Potato virus Y (PVY), and polerovirus Potato leafroll virus (PLRV). PLRV was also detected from individual viruliferous aphids or composites of viruliferous and healthy aphids. PVY and PLRV were detected from extracts immobilized on nitrocellulose membranes, stored for more than 65–273 days at room temperature (25 °C). The protocol was companed with the ‘long protocols’ involving enzyme and phenol extraction for aphids or sodium sulfite and phenol extraction for tubers. The simplified protocol was found comparable in sensitivity to these long procedures, and is especially suitable for those regions where specialized PCR laboratories are only available in centralized locations. Viral RNA immobilized membranes can be mailed out for detection by RT–PCR to these centralized laboratories from remote areas of the country.

Vegetatively propagated plants free from virus- or viroid-induced diseases are integral of the success of many agricultural and horticultural sectors. Reverse transcription – polymerase chain reaction (RT–PCR) has emerged as an effective method to detect the presence of causal agents and has been used as a management strategy to ensure virus- or viroid-free propagation. Advances in understanding virus and viroid genome structures and the availability of genomic sequences of viruses and viroids (and their strains) have made it possible to create primers for specific RT– PCR. This has permitted RT–PCR to evolve into a technique that can simultaneously detect multiple pathogens in a single sample. Multiplex RT–PCR can reduce cost and save time. A further modification of multipex RT–PCR, termed competitive RT–PCR, with a single-antisense and multiple-sense primers, has been developed. It facilitates simultaneous identification and differentiation of a group of virus strains. The methodology of multiplex RT–PC...

A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVYN) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVYNTN) (Tu 660) were tested for their phenotypes by inoculation to potato plants of three potato cultivars. Upon inoculation with Tu 660, tubers of the cultivars ‘Norchip’ and ‘Ranger Russet’ developed potato tuber necrotic ringspot disease (PTNRD) but not the tubers of ‘Russet Burbank’. N-Jg failed to induce PTNRD in the tested cultivars. The genomic RNAs of both strains were completely sequenced and analysed. High homology (98% and 99% identity on nucleotide and polyprotein, respectively) was found between Tu 660 and N-Jg. When polyproteins were compared with other isolates, high identity was observed between Tu 660 and an European (Eu) PVYN-605 (98%) and with an Eu-PVYNTN-H (96%). However, when individual mature proteins were compared, much lower identities (86.5–94%) were found between Tu 660 and PVYNTN-H compared to 98–99.5% between Tu 660 and PVYN-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVYNTN-H is a hybrid molecule of the genomic RNA recombination of PVYO and Eu-PVYN as shown by Glais et al. (Arch Virol 147, 363–378), whereas NA-PVYNTN Tu 660 is free of recombination points. Phylogenetic analysis confirmed this observation, and suggested that, in light of high homology, the Tu 660 might have evolved from NA-PVYN by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVYNTN Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.

Two strains of Potato virus Y (PVY), the common (PVY O ) and the tobacco veinal necrosis (PVY N ) have been known for decades. More recently, a tuber ringspot necrosis (PVY NTN ), and several recombinants of PVY O and PVY N (designated here as PVY N:O ) have been described. Further, the PVY N group of strains have been assigned to two geographical subgroups of European (EU) PVY N/NTN and the North American (NA) PVY N/NTN . The evolution of new PVY N strains, has complicated the diagnosis, which requires a combination of bioassay, serological and molecular assays. To simplify the identification and differentiation of various PVY N strain groups, a competitive (single antisense and multiple sense primers) reverse transcription-polymerase chain reaction (RT-PCR) was used, making use of minor differences in the variable region part of the PVY genome. Specifically, primers based on small variations in nucleotide stretches of P1 gene permitted a broad range separation of PVY O and PVY N groups and the specific detection of strain subgroups. The primer pairs designed for identifying PVY O , EU-PVY N/NTN , NA-PVY N and NA-PVY NTN are described. Primer pairs can be used in a uniplex (single pair of primer) or multiplex (duplex, tetraplex or pentaplex) competitive RT-PCR, allowing simultaneous testing for any combination of PVY O , EU-PVY N/NTN , NA-PVY N and NA-PVY NTN .

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

In 1960s, potato spindle tuber was thought to be a viral disease. In 1971, the agent of the disease was characterised as a low-molecular weight infectious ribonucleic acid (RNA), which was named as ‘viroid’, specifically Potato spindle tuber viroid (PSTVd)). Since then, more than 30 plant diseases in horticultural and ornamental plants have been shown to be caused by different viroids globally. Viroids are single-stranded RNA, covalently closed circular molecule, without any protein coat. They are the smallest known plant pathogen containing RNA genome ranging from 246 nucleotides (Coconut cadang–cadang viroid) to 399 nucleotides (Chrysanthemum chlorotic mottle viroid). Some viroids are located in the plant cell nucleus (pospiviroids) and others in the chloroplast (avsunviroids). With the recognition of pathogenic nature of viroid, specific detection methodologies were developed, which enabled detection of PSTVd in seed-potato tubers prior to their planting in the field, and thus PSTVd was prevented from spreading the disease. As a result, PSTVd was eradicated from Canada in late 1980s. Viroids similar to PSTVd (Pospiviroid) have been discovered and they are detected in symptomless ornamental plants. Although, PSTVd has been eradicated from Canada, there is a strong possibility of viroid introduction from other plants besides potato and tomato and causing PSTVd like diseases.