Potato Leafroll Virus Infection Research Articles

A new simple and effective method for PLRV infection to screen for virus resistance in potato

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

Knockdown of capsid protein encoding novel ATPase domain inhibits genome packaging in potato leafroll virus.

Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.

Incidence and molecular characterization of potato leaf roll virus in seed potato production in Serbia

The distribution and frequency of potato leaf roll virus in the four most important potato growing regions in Serbia were studied during the seven years (2012–18). One hundred randomly collected potato tubers were sampled from each seed lot. The young leaves that developed in three weeks were sampled and tested to record infection rate. The presence of potato leaf roll virus was detected by double-antibody sandwich enzyme-linked immunosorbent assay and disease incidence was calculated using standard formula. The obtained result showed that the highest prevalence of potato leaf roll virus was detected from seed potato samples originated from the Raski region during 2018 (20.7%), while in the Moravicki region, only 2.3–11.1% of the potato leaf roll virus was detected every year. The average annual potato leaf roll virus infection was the highest in 2012 (8.4%) and 2018 (8.0%). For further confirmation of potato leaf roll virus infection, reverse transcription (RT)-PCR was performed using specific primers PLRVCPvEcoRI /PLRVCPcNcoI, designed to amplify a 650 bp fragment of the full-length coat protein gene. The PCR products derived from 26 isolates were directly sequenced using the same primer pair as in RT-PCR. The coat protein sequence analysis revealed that the Serbian potato leaf roll virus isolates showed very low nucleotide diversity (95.9–100%). They shared the highest nt identities of 98.08–99.36% with the sequences of potato leaf roll virus isolates deposited in the GenBank from other parts of the world. Phylogenetic analysis and the haplotype network of the coat protein gene sequences showed that the Serbian potato leaf roll virus isolates could be classified in two different groups indicating two possible introductions of the virus to Serbia. The results of this study confirmed the importance of potato leaf roll virus in seed potato production in Serbia. Additionally, this research highlights the need for a continuous monitoring of the potato seeds produced in Serbia as well as imported seeds for the presence of potato leaf roll virus.

Direct and Indirect Effect via Endophytism of Entomopathogenic Fungi on the Fitness of Myzus persicae and Its Ability to Spread PLRV on Tobacco.

Simple SummaryAphids are major crop pests that are feeding on plant sap and transmitting plant viruses, thus inducing high yield losses worldwide. As chemical pesticides are decreasingly used in plant protection, fungi that cause disease to insects (entomopathogenic fungi) are one of the promising alternatives. They are commonly applied by spraying plants to protect them against herbivores. When applied, some fungi penetrate and live within plant tissues, thus helping to internally protect from insect attacks and other plant diseases. The aim of our study was to assess the effects of entomopathogenic fungi (EPF) applied firstly by contact after insect direct spraying, secondly by endophytic plant inoculation, and thirdly by associated both methods assessing the green peach aphid performances. The impact of the presence of endophytic entomopathogenic fungi (EEPF) in plant tissues on virus transmission by aphids was also considered. We found that the EPF Beauveria bassiana killed the green peach aphid and reduced its fecundity regardless of the application method. On fungal-inoculated plants, there was also a high mortality of aphid nymphs and infection by the potato leafroll virus (PLRV) was delayed by about a week with the EEPF treatment compared to fungal-free plants. This study showed that spraying plant leaves with EPF not only has a direct insecticidal effect against insects but could also have beneficial side effects for the plant against viruses.Aphids are major crop pests that transmit more than half of all insect-vectored plant viruses responsible for high yield losses worldwide. Entomopathogenic fungi (EPF) are biological control agents mainly used by foliar application to control herbivores, including sap-sucking pests such as aphids. Their ability to colonize plant tissues and to interact with diverse plant pathogenic microorganisms have been reported. In our study, we evaluated the effectiveness of Beauveria bassiana ((Balsamo-Crivelli) Vuillemin) directly applied by contact or/and indirectly via endophytism in tobacco plants (Nicotiana tabacum L.) against the virus vector Myzus persicae (Sulzer) carrying the Potato leafroll virus (PLRV) or not. We found that both contact treatment and endophytic colonization of leaves significantly increased aphid mortality and decreased the fecundity rate when compared to control plants. In addition, on fungal-colonized leaves, viruliferous aphids were more negatively impacted than virus-free ones and nymph mortality was significantly higher than on fungal-free plants. Furthermore, we assessed PLRV transmission by M. persicae on tobacco plants inoculated with either B. bassiana or Metarhizium acridum ((Driver and Milner) JF Bischoff, Rehner, and Humber) as source or/and recipient plants. Myzus persicae was found to acquire and transmit PLRV regardless of the treatment. Nevertheless, the infection rate of endophytically colonized plants was lower at a seven-day incubation period and had increased to almost 100% after fifteen days. These results suggest that B. bassiana is effective against aphids, both by contact and via endophytism, and both B. bassiana and M. acridum delayed PLRV infection in tobacco.

A polerovirus, Potato leafroll virus, alters plant-vector interactions using three viral proteins.

Potato leafroll virus (PLRV), genus Polerovirus, family Luteoviridae, is a major pathogen of potato worldwide. PLRV is transmitted among host plants by aphids in a circulative-nonpropagative manner. Previous studies have demonstrated that PLRV infection increases aphid fecundity on, and attraction to, infected plants as compared to controls. However, the molecular mechanisms mediating this relationship are still poorly understood. In this study, we measured the impact of PLRV infection on plant-aphid interactions and plant chemistry in two hosts: Solanum tuberosum and Nicotiana benthamiana. Our study demonstrates that PLRV infection attenuates the induction of aphid-induced jasmonic acid and ethylene in S. tuberosum and N. benthamiana. Using transient expression experiments, insect bioassays and chemical analysis, we show that expression of three PLRV proteins (P0, P1, and P7) mediate changes in plant-aphid interactions and inhibition of aphid-induced jasmonic acid and ethylene in N. benthamiana. This study enhances our understanding of the plant-vector-pathogen interface by elucidating new mechanisms by which plant viruses transmitted in a circulative manner can manipulate plant hosts.

Molecular, serological and biological characterizations of Potato Leaf Roll Virus in infected potato plants in Egypt, and its effects on plant cell organelles

Potato Leaf Roll Virus (PLRV) is one of the most serious viruses infecting potato plant (Solanum tuberosum L.) in Egypt. Indirect Enzyme Linked Immunosorbent Assay (Indirect- ELISA) results revealed that 70 % of the collected samples were infected with PLRV. A multiplex Polymerase Chain Reaction (PCR) was carried out using three different sets of primers, specific for both PLRV and Potato virus Y (PVY) isolates. For confirmation; the movement coat protein (MP) gene was isolated from the infected plant tissues, and a band with molecular size 336 bp was obtained using Reverse transcription-Polymerase chain reaction (RT-PCR). The DNA sequence of the Egyptian PLRV-Banha -MP gene was deposited in GenBank under an accession number of KR002119. Moreover, sequence analysis revealed that the Egyptian PLRV isolate was closely related to a New Zealand isolate of PLRV (GU002341), with identity of 100%. Transmission electron microscope (TEM) examination of PLRV showed isometric particles, with approximate size of 24-30 nm. The cytopathological examination of the potato plant infected with PLRV revealed many cellular effects such as; partially degraded and deformed chloroplast, starch with an increased size and color change, in addition to nucleus and cytoplasmic bridge. It could be concluded that PLRV is present in Egypt, infecting most of the potato cultivars. Moreover, four different strains of PLRV were detected based on the Single strand conformation polymorphism (SSCP) of the MP gene. The aims of the current study were to identify the PLRV infection of potato plant in Egypt using; molecular, serological and biological methods, in addition to studying the effect of this virus on potato cell organelles. This is the first record of the presence of four different strains of PLRV infecting potato in Egypt, using SSCP assay.

Disruption of Chloroplast Function Through Downregulation of Phytoene Desaturase Enhances the Systemic Accumulation of an Aphid-Borne, Phloem-Restricted Virus.

Chloroplasts play a central role in pathogen defense in plants. However, most studies explaining the relationship between pathogens and chloroplasts have focused on pathogens that infect mesophyll cells. In contrast, the family Luteoviridae includes RNA viruses that replicate and traffic exclusively in the phloem. Recently, our lab has shown that Potato leafroll virus (PLRV), the type species in the genus Polerovirus, forms an extensive interaction network with chloroplast-localized proteins that is partially dependent on the PLRV capsid readthrough domain (RTD). In this study, we used virus-induced gene silencing to disrupt chloroplast function and assess the effects on PLRV accumulation in two host species. Silencing of phytoene desaturase (PDS), a key enzyme in carotenoid, chlorophyll, and gibberellic acid (GA) biosynthesis, resulted in a substantial increase in the systemic accumulation of PLRV. This increased accumulation was attenuated when plants were infected with a viral mutant that does not express the RTD. Application of GA partially suppressed the increase in virus accumulation in PDS-silenced plants, suggesting that GA signaling also plays a role in limiting PLRV infection. In addition, the fecundity of the aphid vector of PLRV was increased when fed on PDS-silenced plants relative to PLRV-infected plants.

Temperature and CO2 Level Influence Potato leafroll virus Infection in Solanum tuberosum.

We determined the effects of atmospheric temperature (10–30 ± 2°C in 5°C increments) and carbon dioxide (CO2) levels (400 ± 50 ppm, 540 ± 50 ppm, and 940 ± 50 ppm) on the infection of Solanum tuberosum cv. Chubaek by Potato leafroll virus (PLRV). Below CO2 levels of 400 ± 50 ppm, the PLRV infection rate and RNA content in plant tissues increased as the temperature increased to 20 ± 2°C, but declined at higher temperatures. At high CO2 levels (940 ± 50 ppm), more plants were infected by PLRV at 30 ± 2°C than at 20 or 25 ± 2°C, whereas PLRV RNA content was unchanged in the 20–30 ± 2°C temperature range. The effects of atmospheric CO2 concentration on the acquisition of PLRV by Myzus persicae and accumulation of PLRV RNA in plant tissues were investigated using a growth chamber at 20 ± 2°C. The M. persicae PLRV RNA content slightly increased at elevated CO2 levels (940 ± 50 ppm), but this increase was not statistically significant. Transmission rates of PLRV by Physalis floridana increased as CO2 concentration increased. More PLRV RNA accumulated in potato plants maintained at 540 or 940 ± 50 ppm CO2, than in plants maintained at 400 ± 50 ppm. This is the first evidence of greater PLRV RNA accumulation and larger numbers of S. tuberosum plants infected by PLRV under conditions of combined high CO2 levels (940 ± 50 ppm) and high temperature (30 ± 2°C).

Responses of Aphid Vectors of Potato leaf roll virus to Potato Varieties.

Potato leaf roll virus (PLRV) can reduce tuber yield and quality in potato. Green peach aphid (Myzus persicae [Sulzer]) and potato aphid (Macrosiphum euphorbiae [Thomas]) are the two most important potato-colonizing PLRV vectors in the Pacific Northwest. We compared My. persicae and Ma. euphorbiae densities and PLRV incidences among potato varieties in the field to clarify the relationships between aphid abundance and PLRV incidence in plants. Aphids were sampled weekly over three years in the potato varieties Russet Burbank, Ranger Russet, and Russet Norkotah in a replicated field trial. In all years, My. persicae was more abundant than Ma. euphorbiae, representing at least 97% of samples. My. persicae densities did not differ among potato varieties across years; very low numbers of Ma. euphorbiae precluded such statistical comparisons for this species. PLRV infection did not differ significantly among potato varieties, although the percent of PLRV-infected plants differed among years when all varieties were combined (46% in 2013, 29% in 2011, 13% in 2012). For Ranger Russet and Russet Norkotah, PLRV incidence was positively correlated with aphid abundance as well as proportion of PLRV-positive aphids. In Russet Burbank, only aphid abundance was positively correlated with PLRV infection. Our results suggest that the three most commonly grown potato varieties in our region do not differ in their susceptibility to PLRV infection, and that aphid density was a consistent indicator of the risk of infection by this virus across varieties. Both of these findings can be used to hone PLRV monitoring and modeling efforts.

The Effects of High Temperature on Infection by Potato virus Y, Potato virus A, and Potato leafroll virus.

We examined the effects of temperature on acquisition of Potato virus Y-O (PVY-O), Potato virus A (PVA), and Potato leafroll virus (PLRV) by Myzus persicae by performing transmission tests with aphids that acquired each virus at different temperatures. Infection by PVY-O/PVA and PLRV increased with increasing plant temperature in Nicotiana benthamiana and Physalis floridana, respectively, after being transmitted by aphids that acquired them within a temperature range of 10–20°C. However, infection rates subsequently decreased. Direct qRT-PCR of RNA extracted from a single aphid showed that PLRV infection increased in the 10–20°C range, but this trend also declined shortly thereafter. We examined the effect of temperature on establishment of virus infection. The greatest number of plants became infected when N. benthamiana was held at 20°C after inoculation with PVY-O or PVA. The largest number of P. floridana plants became infected with PLRV when the plants were maintained at 25°C. PLRV levels were highest in P. floridana kept at 20–25°C. These results indicate that the optimum temperatures for proliferation of PVY-O/PVA and PLRV differed. Western blot analysis showed that accumulations of PVY-O and PVA coat proteins (CPs) were lower at 10°C or 15°C than at 20°C during early infection. However, accumulation increased over time. At 25°C or 30°C, the CPs of both viruses accumulated during early infection but disappeared as time passed. Our results suggest that symptom attenuation and reduction of PVY-O and PVA CP accumulation at higher temperatures appear to be attributable to increased RNA silencing.

Examination of an isolate of Potato leaf roll virus that does not induce visible symptoms in the greenhouse

Over the last 30 years the importance of Potato leaf roll virus (PLRV) in commercial potato and seed potato production has decreased considerably. Since PLRV is transmitted by aphids in a persistent manner it can be controlled by applying a systemic insecticide. However, the development of insecticide resistance in the main vectors of PLRV Myzus persicae, Aulacorthum solani, Rhopalosiphoninus latysiphon, Aphis fabae, A. nasturtii, A. frangulae and Macrosiphum euphorbiae, and the development of isolates of PLRV that do not induce visible symptoms in some potato cultivars may lead to a resurgence in the significance of PLRV. Isolates of this type were found repeatedly during growing-on tests in Lower Saxony, Germany. In this study we examined such a symptomless isolate. The visible symptoms induced by this isolate in different potato cultivars were compared with those induced by isolates causing typical symptoms of a PLRV infection. By using quantitative real-time PCR the quantifiable amount of viral RNA was determined. Under climate chamber conditions all the isolates tested induced similar symptoms and did not differ in viral RNA content. Complete sequences for the tested isolates were obtained and used in a phylogenetic analysis. All the PLRV isolates compared were very similar at the molecular level. Several motifs that could play a role in symptom expression were analyzed, but none of them were correlated with the absence of symptoms in potato plants during growing-on tests. The discrepancy between the observations recorded in the growing-on tests and our experiments are discussed.

Development of Molecular Markers Closely Linked to the Potato Leafroll Virus Resistance Gene, Rlr etb , for use in Marker-Assisted Selection

Potato leafroll virus (PLRV) is a major pathogen of potato with worldwide impact on seed and commercial production of potato. In North America, the primary potato cultivars grown by industry are not resistant to PLRV and require the application of insecticides to control the aphid vector of PLRV, so as to prevent PLRV infection of the crop. The Solanum etuberosum- derived dominant gene Rlretb confers resistance to PLRV and has been mapped to chromosome 4. The previous closest marker to Rlretb, C2_At1g42990, was mapped 13.6 cM from the gene in a BC3 population. Cleaved amplified polymorphic sequence markers were developed in the region surrounding C2_At1g42990 and mapped along with Rlretb in 102 BC4 progeny. Sixteen markers were identified surrounding Rlretb, with flanking markers at 2.1 and 9.3 cM. Two markers, 1367-8a and DMB32-11, both 2.1 cM from Rlretb, are shown to be well suited for marker assisted selection. The development of additional molecular markers more closely linked to Rlretb will greatly facilitate its use in potato breeding and its continued introgression into cultivated potato for the development of PLRV-resistant cultivars.

Amino Acid Sequence Motifs Essential for P0-Mediated Suppression of RNA Silencing in an Isolate of Potato leafroll virus from Inner Mongolia

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Different Reactions of Potato Varieties to Infection by Potato Leafroll Virus, and Associated Responses by its Vector, Myzus persicae (Sulzer)

Vector-dependent plant pathogens can alter their hosts such that vector behavior and pathogen spread are affected. For example, Potato leafroll virus (PLRV)-induces changes in volatiles emitted by potato plants (Solanum tuberosum L.) that influence settling behavior by Myzus persicae, a principal vector of the virus. Prior work in this pathosystem has utilized a single potato variety, Russet Burbank, but as is true for other plant responses to biotic stresses, responses may differ among plant genotypes. To examine this, PLRV-induced changes in headspace volatile organic compounds (VOCs) and associated aphid responses to these VOCs were compared among four potato varieties (Chipeta, Desiree, IdaRose, and Russet Burbank). Total headspace VOCs differed among the varieties and were differentially induced by PLRV infection such that the effect of variety, infection, and their interaction was significant; two of the varieties had increased concentrations of headspace VOCs, and two did not. MANOVA for the effect of infection and variety on total VOCs and major VOC classes also was significant. A principal component analysis (PCA) partially separated the VOC profiles from the four varieties. Aphid arrestment differed in response to the VOCs of the four varieties, and was greater on those that were more readily infected by PLRV (Desiree and Russet Burbank) as compared with those that were less readily infected (Chipeta and IdaRose). Aphid responses were not clearly related to specific characteristics of blends, such that total VOCs and composition appear to contribute. The four varieties used in this study have distinct pedigrees representative of different cultivated forms of S. tuberosum. Although cultivated potato varieties have been subjected to genetic manipulation by humans, the differences in PLRV induced changes in VOCs nonetheless indicate the potential for complex effects of PLRV infection on host plant VOC emissions, and vector responses in managed, and natural systems.

The effect of an alternate weed host, hairy nightshade, Solanum sarrachoides (Sendtner) on green peach aphid distribution and Potato leafroll virus incidence in potato fields of the Pacific Northwest

Hairy nightshade, Solanum sarrachoides (Sendtner) is an annual solanaceous weed prevalent in potato farmlands of the Pacific Northwest. S. sarrachoides is also a reservoir for Potato leafroll virus (PLRV) and its most important vector, the green peach aphid, Myzus persicae (Sulzer). Green peach aphids prefer S. sarrachoides than potato, Solanum tuberosum (L.), and produce more nymphs on S. sarrachoides than on potato. Increased green peach aphid preference for S. sarrachoides and greater fecundity on this plant could lead to overcrowding and movement toward neighboring potato plants. To test our hypothesis, field trials were conducted at Kimberly, Idaho during the summers of 2003 and 2004. A row of S. sarrachoides seedlings was planted in the middle of potato plots and naturally occurring green peach aphid populations were monitored weekly on potato plots with and without S. sarrachoides. More aphids were consistently found on S sarrachoides plants than on potato plants. More aphids were found on potato plants in plots with S. sarrachoides plants than without S. sarrachoides. Targeted insecticide applications on S. sarrachoides rows alone reduced the number of aphids on potato plants in those plots, suggesting that insecticide sprays prevented aphid movement from S. sarrachoides to potato. Testing of potato tuber sprouts 90 days after harvest by double antibody sandwich ELISA indicated a greater PLRV infection rate on plots with S. sarrachoides than plots without S. sarrachoides. Therefore, the presence of S. sarrachoides in potato plots will likely increase aphid populations and PLRV incidence on neighboring potato plants.

First Report of Tomato chlorosis virus in Potato in Brazil.

Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system.

Hairy Nightshade as a Potential Potato leafroll virus (Luteoviridae: Polerovirus) Inoculum Source in Pacific Northwest Potato Ecosystems

Hairy nightshade, Solanum sarrachoides, is a solanaceous weed found abundantly in Pacific Northwest potato ecosystems. It serves as a reservoir for one of the important potato viruses, Potato leafroll virus (PLRV) (Luteoviridae: Polerovirus), and its most important vector, the green peach aphid, Myzus persicae (Homoptera: Aphididae). Laboratory research indicated an increased green peach aphid settling and performance on S. sarrachoides than on potato. It also revealed that green peach aphids transmitted PLRV more efficiently from S. sarrachoides to potato than from potato to potato. To test the efficiency of S. sarrachoides as an inoculum source in the field, a two season (2004 and 2005) trial was conducted at Kimberly, Idaho. Two inoculum sources, PLRV-infected potato and PLRV-infected S. sarrachoides, were compared in this trial. Green peach aphid density and temporal and spatial PLRV spread were monitored at weekly intervals. Higher densities of green peach aphids were observed on plots with S. sarrachoides and inoculum sources (PLRV-infected S. sarrachoides and potato) than on plots without S. sarrachoides and inoculum sources. PLRV infection in plots with PLRV-infected S. sarrachoides was similar to or slightly higher than in plots with PLRV-infected potato as an inoculum source. Temporal and spatial PLRV spread was similar in plots with either inoculum source. Thus, S. sarrachoides is as efficient as or a better PLRV inoculum source than potato.

Mechanism of strong resistance to Potato leafroll virus infection in a clone of potato (Solanum tuberosum)

AbstractThe Solanum tuberosum clone G8107(1) is highly resistant to Potato leafroll virus (PLRV) accumulation following graft inoculation and to PLRV infection by aphids. In glasshouse/insectary experiments, plants were inoculated with viruliferous aphids (Myzus persicae); infection was detectable in some inoculated leaves (by enzyme‐linked immunosorbent assay or reverse transcription polymerase chain reaction) but not in petioles, stems or non‐inoculated leaves of inoculated plants. Secondary infection in tuber‐progeny plants of inoculated plants was rare in G8107(1) (detected in only one of 86 tuber‐progeny plants of 24 inoculated plants), while universal in susceptible controls. This resistance did not break down when tuber sprouts were inoculated or when a more efficient M. persicae vector clone was used. Aphids were able to acquire and transmit PLRV infection from infected plants of G8107(1) to test plants but at a lower frequency than from the susceptible cv. Maris Piper. We suggest that an important element of the very strong resistance of this clone to PLRV infection is resistance to virus movement within leaves or from leaves to petioles, thus preventing spread to the rest of the plant.

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

BackgroundThe green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species.ResultsSequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes.ConclusionNew genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects.

Utilization of Shoot Cuttings for Elimination of PLRV and PVY by Thermotherapy and Chemotherapy from Potato (Solanum tuberosum L.)

Heat and ribavirin treatments were applied for the elimination of potato leaf roll virus (PLRV) and potato virus Y (PVY) from the potato genotype F 9-99. The explants, rooted young plantlets, cultured on MS medium with and without ribavirin (20 mg l<sup>-1</sup>) were subjected to thermotherapy (35ºC/31ºC, 4 h alternating periods) and room temperature (25ºC) for 30 days. Double antibody sandwich - enzyme linked immunosorbent assay (DAS-ELISA) testing following the therapies revealed that ribavirin alone was enable to eliminate 10% each of PLRV and PVY, whereas along with thermotherapy its efficacy increased to 25% PLRV and 20% PVY elimination. In another experiment, three potato genotypes F 9-99 infected with PLRV and PVY, Gui Valley with PLRV infection and Rose Valley with PVY infection were evaluated. Ribavirin (20 mg l<sup>-1</sup>) and ASA (acetylsalicylic acid) (10<sup>-5</sup> M) were supplemented in liquid culture medium alone or in combination with thermotherapy for three successive cycles of 30-36 days interval. Heat and/or ribavirin suppressed survival and plant height whereas ASA promoted the survival as well as plant height even under heat treatment. After each cycle, the effect of treatment on virus elimination was evaluated by DAS-ELISA. The combined application of ribavirin and ASA with thermotherapy after three cycles of treatment showed up to 47.4% PLRV and 57.9% PVY elimination in F 9-99. In the case of single viral infection of PLRV or PVY the same combined treatment showed up to 58.8% PLRV and 61.1% PVY free plantlets. Virus elimination was also confirmed by transplanting in vitro grown plantlets in a net house and then retesting after 45 days of in vivo growth. This novel technique would be highly efficient for virus elimination within a short duration in diverse genotypes of potato. <i>Nepal Journal of Science and Technology </i> 7 2006