Simulation modelling of potato virus Y spread in relation to initial inoculum and vector activity

Potato virus Y(PVY)is a non-persistent virus that is transmitted by many aphid species and causes significant damage to potato production.We constructed a spatially-explicit model simulating PVY spread in a potato field and used it to investigate possible effects of transmission efficiency,initial inoculum levels,vector behavior,vector abundance,and timing of peak vector activity on PVY incidence at the end of a simulated growing season.Lower PVY incidence in planted seed resulted in lower virus infection at the end of the season.However,when populations of efficient PVY vectors were high,significant PVY spread occurred even when initial virus inoculum was low.Non-colonizing aphids were more important for PVY spread compared to colonizing aphids,particularly at high densities.An early-season peak in the numbers of noncolonizing aphids resulted in the highest number of infected plants in the end of the season,while mid-and late-season peaks caused relatively little virus spread.Our results highlight the importance of integrating different techniques to prevent the number of PVY-infected plants from exceeding economically acceptable levels instead of trying to control aphids within potato fields.Such management plans should be implemented very early in a growing season.

Three sensitive and reliable serological assays for detection of potato virus A in potato plants

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A(PVA)is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies(MAbs)(2D4,8E11,14A6 and 16H10)using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680(w/v,g m L^(-1))by ACP-ELISA or up to1:10240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

Screening and Identification of Tobacco Mutants Resistant to Potato Virus Y( PVY)

[ Objective] This study aimed to screen and identify tobacco mutants resistant to potato virus Y （PVY）, thus laying the foundation for obtaining PVY resistance genes. [ Method ] At seedling stage, tobacco mutant materials were inoculated with PVY virus and preliminarily screened by naked-eye observation. Enzyme-linked immunosorbent assay （ELISA）, real-time fluorescence quantitative PCR and test strip assay were performed to further identify the pre-screened PVY- resistant seedlings. [ Result ] In 2011, two highly PVY-resistant tobacco mutants （MZE2-15 and MZE2-16） and three PVY-tolerant mutants （MZE2-70, MZE2- 207 and MZE2-228） were obtained, which were further screened and identified in 2012. According to the results, tobacco mutant materials MZE2-407 and MZE2- 428 were susceptible to PVY; mutant materials MZE2-16 and MZE2-15 were resistant to PVY. [ Conclusion] This study provide theoretical basis for the control of tobacco PVY disease.

Effects of Synergism Between PVY and PVX on Virus Titer and Cell Ultrastructure in Tobacco Plants

The viruses titer and the ultrastructure of infected cells in tobacco host (Nicotiana tabacum cv. Samsun), which doubly infected with potato virus Y necrosis strain (PVYN) and potato virus X (PVX), were studied under greenhouse conditions. The results indicated that PVYN and PVX interacted synergistically. and tobacco plants which doubly infected with PVX and PVYN could greatly increase symptom severity as compared with that induced by the single virus. As determined by triple antibody sandwich enzyme-linked immunosor-bent assay (TAS-ELISA), the titer of PVX in the tobacco leaves infected with both PVYN and PVX was up to 9.10 times higher than the plants infected with PVX only. No significant differences in PVYN titer were detected between singly and doubly infected plants. The enhancement of PVX titer in doubly infected plants was evident in greenhouse and was independent of the virus strains, tested seasons, intervals between PVYN and PVX inoculation. When sections of doubly infected leaves were examined with an electron microscope, it could be frequently found that cells contained pinwheels and large masses of PVX-like particles, pinwheels and laminate inclusions, or all three structures, most of them were swollen, and their chloroplast and other organella were destructed heavily. This suggested that doubly infected cells were involved in the enhancement phenomenon, which seemed to be the result of an increase in the amount of PVX synthesized in them.

Evaluation of potential RNA-interference-target genes to control cotton mealybug, Phenacoccus solenopsis （Hemiptera： Pseudococcuidae）

RNA interference （RNAi） of vital insect genes is a potential tool for targeted pest control. However, selection of the right target genes is a challenge because the RNAi efficacy is known to vary among insect species. Cotton mealybug, Phenacoccus solenopsis, is a phloem-feeding economically important crop pest. We evaluated the RNAi of 2 vital genes, Bursicon （PsBur） and V-ATPase （Ps V-ATPase） as potential targets in P. solenopsis for its control. PCR fragments of PsBur and PsV-ATPase were amplified using cDNA synthesized from the total RNA. The PCR amplicons were cloned into Potato virus X （PVX） to develop recombinant PVX for the inoculation ofNicotiana tabacum plants for bioassays with healthy P. solenopsis. Reverse-transcription-polymerase chain reaction （RT- PCR） was used to validate the expression oftransgenes in the recombinant-PVX-inoculated plants （treated）, and suppression of the target genes in the mealybugs exposed to them. The RT-PCR confirmed the expression of transgenes in the treated plants. Mealybug individuals on treated plants either died or showed physical deformities. Further, the population of mealybug was significantly reduced by feeding on N. tabacum expressing RNAi triggers against PsBur and Ps V-A TPase. The results conclude that RNAi is activated in P. solenopsis by feeding on N. tabacum expressing RNAi triggering elements of PsBur and Ps V-ATPase genes through recombinant PVX vector. Further, V-ATPase and Bursicon genes are potential targets for RNAi-mediated control ofP. solenopsis.

A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunit （rbcS） gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena, transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.