Development of a Simple Dipstick Assay for Operational Monitoring of DDT.

BACKGROUND: Indoor residual spraying (IRS) of DDT is used to control visceral leishmaniasis (VL) in India. However, the quality of spraying is severely compromised by a lack of affordable field assays to monitor target doses of insecticide. Our aim was to develop a simple DDT insecticide quantification kit (IQK) for monitoring DDT levels in an operational setting. METHODOLOGY/ PRINCIPLE FINDINGS: DDT quantification was based on the stoichiometric release of chloride from DDT by alkaline hydrolysis and detection of the released ion using Quantab chloride detection strips. The assay was specific for insecticidal p,p`-DDT (LoQ = 0.082 g/m2). Bostik discs were effective in post spray wall sampling, extracting 25-70% of active ingredient depending on surface. Residual DDT was sampled from walls in Bihar state in India using Bostik adhesive discs and DDT concentrations (g p,p`-DDT/m2) were determined using IQK and HPLC (n = 1964 field samples). Analysis of 161 Bostik samples (pooled sample pairs) by IQK and HPLC produced excellent correlation (R2 = 0.96; Bland-Altman bias = -0.0038). IQK analysis of the remaining field samples matched HPLC data in identifying households that had been under sprayed, in range or over sprayed. INTERPRETATION: A simple dipstick assay has been developed for monitoring DDT spraying that gives comparable results to HPLC. By making laboratory-based analysis of DDT dosing accessible to field operatives, routine monitoring of DDT levels can be promoted in low- and middle- income countries to maximise the effectiveness of IRS.

λ-Carrageenan Suppresses Tomato Chlorotic Dwarf Viroid (TCDVd) Replication and Symptom Expression in Tomatoes.

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(É©)-, 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.

Adverse Effects of UV-B Radiation on Plants Growing at Schirmacher Oasis, East Antarctica.

This study aimed to assess the impacts of ultraviolet-B (UV-B) radiation over a 28-day period on the levels of pigments of Umbilicaria aprina and Bryum argenteum growing in field. The depletion of stratospheric ozone is most prominent over Antarctica, which receives more UV-B radiation than most other parts of the planet. Although UV-B radiation adversely affects all flora, Antarctic plants are better equipped to survive the damaging effects of UV-B owing to defenses provided by UV-B absorbing compounds and other screening pigments. The UV-B radiations and daily average ozone values were measured by sun photometer and the photosynthetic pigments were analyzed by the standard spectrophotometric methods of exposed and unexposed selected plants. The daily average atmospheric ozone values were recorded from 5 January to 2 February 2008. The maximum daily average for ozone (310.7 Dobson Units (DU)) was recorded on 10 January 2008. On that day, average UV-B spectral irradiances were 0.016, 0.071, and 0.186 W m(-2) at wavelengths of 305, 312, and 320 nm, respectively. The minimum daily average ozone value (278.6 DU) was recorded on 31 January 2008. On that day, average UV-B spectral irradiances were 0.018, 0.085, and 0.210 W m(-2) at wavelengths of 305, 312, and 320 nm, respectively. Our results concludes that following prolonged UV-B exposure, total chlorophyll levels decreased gradually in both species, whereas levels of UV-B absorbing compounds, phenolics, and carotenoids gradually increased.

The discovery and eradication of potato spindle tuber viroid in Canada.

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.

Retrospective Quarterly Cohort Monitoring for patients with Visceral Leishmaniasis in the Indian subcontinent: outcomes of a pilot project.

OBJECTIVE: To evaluate a new tool for the monitoring of Visceral Leishmaniasis (VL) treatment outcomes in primary healthcare (PHC) settings, adapted from the standardised Retrospective Quarterly Cohort Monitoring done in tuberculosis control. METHODS: We developed standard case definitions for early and late VL treatment outcomes, a single register allowing for one-line entry per patient as registration tool, and quarterly reporting formats for the clinical outcomes. We pilot-tested these tools in three Indian Primary Health Centres and two Nepalese district hospitals, as well as in a charity VL treatment centre and a university hospital. RESULTS: Data collection for early treatment outcome was easily implemented but information on late treatment outcome was hard to obtain. Effectiveness of Miltefosine under routine care conditions was about 87% at end of treatment, and 76% at 6 months post-treatment related to the high number of patients lost to follow up at the latter end point. CONCLUSION: A retrospective cohort monitoring methodology is conceptually a good framework for monitoring clinical outcomes for chronic conditions as VL. The monitoring of early outcomes of VL treatment is perfectly feasible in Primary Care settings. The completeness of information on late outcomes can be improved by a number of strategies that remain to be field tested. Generally, clinical outcome monitoring should be strengthened in the VL control programmes.

Recognition and Molecular Discrimination of Severe and Mild PVYO Variants of Potato virus Y in Potato in New Brunswick, Canada.

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.

An alkaline solution simplifies nucleic acid preparation for RT-PCR and infectivity assays of viroids from crude sap and spotted membrane.

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.

Evaluation of a simple membrane-based nucleic acid preparation protocol for RT-PCR detection of potato viruses from aphid and plant tissues.

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 6h. 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 degrees 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.

An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids.

A primer pair for reverse transcription-polymerase chain reaction (RT-PCR), based on the conserved sequences of the members of genus Pospiviroid was designed to yield a fragment of about 200 base pairs (bp). Since pospiviroids infect a large number of plants species and a few members of the genus Pospiviroid have been already detected in some ornamental plants, the primer pair was evaluated for its efficacy using ornamental plants. The method of return-polyacrylamide gel electrophoresis (R-PAGE) was used to determine the general presence of viroids in the test samples. Efficacy of the primer pair for members of genus Pospiviroid was demonstrated by the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in potato, Chrysanthemum stunt viroid and Iresine viroid in Verbena and Vinca species, and Citrus exocortis viroid in Impatiens species. Specificity of the primer pair became evident, where additional viroids were detected by R-PAGE in Coleus and Magilla species, but they were not amplified by the Pospiviroid primer. This primer pair would be of benefit in indexing ornamental plants in quarantine samples or in viroid-free certification schemes, irrespective of their actual identity.

Specific differentiation of recombinant PVY(N:O) and PVY(NTN) isolates by multiplex RT-PCR.

The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVYN) and potato tuber necrotic group (PVY(NTN)) contain segments of the PVYO and the PVY(N) genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVY(NTN) at sites HC/Pro-P3, 6K2-NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVYN isolates (termed PVY(N:O)). Protocols for specific differentiation of the recombinant PVY(NTN) and PVY(N:O) from the non-recombinant PVYN are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641bp from the recombinant PVY(NTN) and PVY(N:O). The second and third primer pairs exclusively amplified fragments of 448 and 290bp, respectively from the recombinant PVY(NTN). In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290bp) were amplified exclusively from the recombinant PVY(NTN), while only one fragment (641bp) was amplified from the PVY(N:O) isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVYO and North American (NA)-PVY(N/NTN). For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity.

Evolution of North American PVY(NTN) strain Tu 660 from local PVY(N) by mutation rather than recombination.

A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVY(N)) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVY(NTN) (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) PVY(N)-605 (98%) and with an Eu-PVY(NTN)-H (96%). However, when individual mature proteins were compared, much lower identities (86.5-94%) were found between Tu 660 and PVY9(NTN))-H compared to 98-99.5% between Tu 660 and PVY(N)-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVY(NTN)-H is a hybrid molecule of the genomic RNA recombination of PVY(O) and Eu-PVY(N) as shown by Glais et al. (Arch Virol 147, 363-378), whereas NA-PVY(NTN) 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-PVY(N) by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVY(NTN) Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.

Molecular characterization and expression analysis of 1-aminocyclopropane-1-carboxylate oxidase homologs from potato under abiotic and biotic stresses.

In this work, we report cloning of two full-length 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNAs (ACO1 and ACO2) from potato (Solanum tuberosum) and their expression in potato tissues. The sequence data indicate that the two cDNAs share a high degree of homology with each other, and with known ACO genes from other plant species, including monocots and dicots. However, these potato genes lack homology at the 5' and 3' ends, despite similarities in their open reading frames and encoded amino acids. Phylogenetic analysis places them in two subfamilies of ACOs. The genes are tissue specific: expression is high in leaves and low in roots and tubers. In sprouts and tubers, ACO1 is induced by heat (40 degrees C) and cold (0 degrees C) stresses, whereas ACO2 is induced only by cold (0 degrees C). ACO1 is markedly induced in leaves by wounding, soil-flooding, and exogenous application of 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, ACO2 induction is lower under these treatments. ACO1 and ACO2 are regulated very differently in potato leaves with respect to senescence. ACO2 expression is unaffected by senescence, whereas that of ACO1 is closely related to the age and senescence in both attached and detached leaves. Exogenous ACC not only induces ACO1, but also accelerates leaf senescence. ACO1 transcripts are induced significantly in leaves, stems, and tubers in the Potato virus A (PVA)-resistant potato cultivar Shepody when graft inoculated with PVA.

A new approach for the simultaneous differentiation of biological and geographical strains of Potato virus Y by uniplex and multiplex RT-PCR.

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).

Probable geographical grouping of PVY(N) and PVY(NTN) based on sequence variation in P1 and 5'-UTR of PVY genome and methods for differentiating North American PVY(NTN).

An increasing number of countries in recent years have reported the occurrence of potato tuber necrotic ringspot disease (PTNRD), caused by tobacco veinal necrosis strain of Potato virus Y (PVY(N)), belonging to the sub-group tuber necrosis (PVY(NTN)). Methods for the differentiation of PVY(NTN), based on primer sequences often detect isolates of European (EU) type but not the North American (NA) type. To resolve this problem, the nucleotide sequence of 5'-untranslated region (5'-UTR) and the P1 gene of 11 isolates of PVY(N) and PVY(NTN) from the European Union and North America was determined. Sequence comparison and phylogenetic analysis of 5'-UTR and P1 region indicated that PVY(N) isolates from the European Union and North America formed their own separate groups. Intra-group sequence identity for all except one was over 98%, as opposed to the inter-group identity of 90%. Additionally, the PVY(NTN) isolates from the European Union and North America clustered with their respective PVY(N) isolates. This indicates a possible evolution of PVY(NTN) isolates from the PVY(N) isolates of a geographical region. With this information of regional relationships of PVY(NTN) and PVY(N) isolates, two approaches were developed based on a competitive RT-PCR and a restriction pattern, for the differentiation of NA-PVY(NTN) from the local PVY(N) and from EU-PVY(NTN). Thus sequencing of the P1 gene and use of competitive RT-PCR approach could be applicable for determining the possible origin of new occurrences of PVY(NTN) from other geographical regions.

Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids.

Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid.

Specific Detection of Potato Virus A in Dormant Tubers by Reverse-Transcription Polymerase Chain Reaction.

A reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for the detection of potato virus A (PVA) in dormant tubers. A 255-bp amplified product was produced using a primer pair from the P1 gene of the PVA genome. The 255-bp product was detected in nucleic acids from leaves, tubers, and purified virions and was specific to PVA as determined by Southern blot tests and detection by a PVA-specific probe. When presented with seven potato virus/strain nucleic acids and a viroid, singly and in mixed infections, the primer pair did not amplify any products. Its specificity to PVA was further demonstrated by RT-PCR detection of PVA from the known mixtures of PVA and potato virus Y samples. PVA was detected in foliage nucleic acids at a dilution of 1:1024-1:4096 and tuber nucleic acids at 1:256-1:1024. It was uniformly present in various parts of the potato tuber. PVA was detected in composite tuber samples containing a ratio of infected to healthy sap of 1:29 and was readily detected in tubers of several cultivars or breeding lines, in dormant as well as in sprouting tubers stored at 20-25°C for 4 months.