Sensitivity of Small RNA-Based Detection of Plant Viruses

Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21-24 nucleotides, the reassembly of which into longer strands in silico allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of Potato virus A (PVA), each carrying a different marker gene, and an infectious clone of Potato virus Y (PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.Peer reviewe

Case study : Planting methods and beneficial substrate microbes effect on the growth of vegetated roof plants in Finland

Vegetated roofs, often called "green roofs", are popular and necessary in urban greening in densely populated areas. Well-functioning vegetated roofs can provide various ecosystem services to urban residents (e.g., stormwater management, air pollution mitigation, and aesthetic value). Plants essentially determine the actualization of the ecosystem services, thus finding effective ways to establish and maintain the roof plants is important. While greenhouse experiments can be better controlled than field experiments, it is critical to test whether results gained in the greenhouse hold in actual roof conditions. Therefore, we investigated the effects of microbial inoculant, plant species, planting method, and their interactions on plant growth and the beneficial microbes in the roof substrate at the initial establishment of vegetated roofs. The selected plants (i.e., Antennaria dioica, Campanula rotundifolia, Fragaria vesca, Geranium sanguineum, Lotus corniculatus, Thymus serpyllum, Trifolium repens, and Viola tricolor) were established using pre-grown vegetation mats, plug plants, and seeds, each with and without the co-inoculation with Rhizophagus irregularis and Bacillus amyloliquefaciens, two plant growth-promoting microbial species. Eventually, only F. vesca, T. serpyllum, T. repens, and V. tricolor were found successfully settled in either of the three planting methods. Dry aboveground plant biomass was measured to assess the effects of co-inoculation on plant growth. R. irregularis colonization level and B. amyloliquefaciens bacterial density were detected from root and substrate samples, respectively. The results indicated that co-inoculation with R. irregularis and B. amyloliquefaciens successfully colonized target plant species and significantly increased the initial growth of the vegetated roof plants by 18-292%. Additionally, the abundance of R. irregularis was affected by plant species (F. vesca > T. serpyllum > T. repens) and planting methods (seed > plug > mat), while the bacterial density of B. amyloliquefaciens was higher in T. repens roots than the other plant species, and was not affected by planning methods. The results suggest that co-inoculating R. irregularis and B. amyloliquefaciens at the installation phase of vegetated roofs could improve microbial settlement and colonization in the substrate, and consequently achieve synergistic effect on plant growth. The study also provides basis and reference for future vegetated roofs research.Peer reviewe

Resistance to potato virus A and potato virus Y in potato cultivars grown in Finland

Phenotypic expression of resistance to potato Y potyvirus (PVY) and potato A potyvirus (PVA) was tested in 24 potato cultivars and an advanced breeding line using graft-inoculation under controlled conditions in the glasshouse. Resistance phenotypes were determined based on symptom expression and systemic infection detected with DAS-ELISA. Tubers were harvested from the PVA-inoculated plants and tested for PVA with ELISA. Sixteen potato cultivars expressed hypersensitive resistance (HR) to the strain group YO of PVY. Ute expressed extreme resistance (ER) to PVY (strain groups YO and YN) and PVA, and eight cultivars (Amazone, Bintje, Fambo, Posmo, Record, Rosamunda, Saturna and Van Gogh) expressed ER to PVA. These cultivars produced no PVA-infected tubers (tubers of Record were not tested). Matilda and Nicola expressed HR to PVA. The tubers of graft-inoculated Matilda produced no PVA-infected shoots, whereas shoots from Nicola tubers developed necrosis and severe mosaic symptoms and were PVA-infected based on results from ELISA. Comparison with purified PVA antigen (using ELISA) indicated that the secondarily infected shoots of the 14 PVA-susceptible cultivars contained 206-804 ng of PVA antigen per gram of leaf tissue

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.Peer reviewe

Sunagoke Moss (Racomitrium japonicum) Used for Greening Roofs Is Severely Damaged by Sclerotium delphinii and Protected by a Putative Bacillus amyloliquefaciens Isolate

Mosses are ecologically important plants also used for greening, gardening, and decorative purposes. Knowledge of the microbial flora associated with mosses is expected to be important for control and preservation of global and local environments. However, the moss-associated microbial flora is often poorly known. Moss-associated fungi and bacteria may promote plant growth and pest control, but they may be alternative hosts for pathogens of vascular plants. In this study, the fungus Sclerotinia delphinii was identified for the first time as a pathogen that causes severe damage to Sunagoke moss (Racomitrium japonicum). This moss is used for greening roofs and walls of buildings in urban environments owing to its notable tolerance of environmental stresses. Inoculation with the S. delphinii strain SR1 of the mono- and dicotyledonous seed plants Hordeum vulgare, Brassica rapa var. pekinensis, Lactuca sativa, and Spinacia oleracea, in addition to the liverwort Marchantia polymorpha and the moss Physcomitrella patens, showed that the fungus has a wide host range. Colonization with SR1 progressed more rapidly in non-vascular than in vascular plant species. Studies with P. patens under controlled conditions showed that SR1 secreted a fluid during colonization. Treatment with the secretion induced production of reactive oxygen species in the moss. Endogenous peroxidase partially inhibited SR1 colonization of P. patens. A bacterial isolate, most likely Bacillus amyloliquefaciens, that coexists with R. japonicum was antagonistic to SR1 growth. Taken together, the present results suggest that fungal colonization of mosses may be prevented by a peroxidase secreted by the moss and an antagonistic bacterium coexisting in the moss habitat. The findings suggest that there is potential to apply biological control measures for protection of mosses against fungal pathogens.Peer reviewe

Viruses and their significance in agricultural and horticultural crops in Finland

This paper reviews the plant viruses and virus vectors that have been detected in agricultural and horticultural crop plants and some weeds in Finland. The historical and current importance of virus diseases and the methods used for controlling them in cereals, potato, berry plants, fruit trees, ornamental plants and vegetables are discussed. Plant viruses have been intensely studied in Finland over 40 years. Up to date, 44 plant virus species have been detected, and many tentatively identified viruses are also reported. Control of many virus diseases has been significantly improved. This has been achieved mainly through changes in cropping systems, production of healthy seed potatoes and healthy stocks of berry plants, fruit trees and ornamental plants in the institutes set up for such production, and improved hygiene. At the present, barley yellow dwarf luteovirus, potato Y potyvirus and potato mop-top furovirus are considred to be economically the most harmful plant viruses in Finland

Phenotyping viral infection in sweetpotato using a high-throughput chlorophyll fluorescence and thermal imaging platform

Background Virus diseases caused by co-infection with Sweet potato feathery mottle virus (SPFMV) and Sweetpotato chlorotic stunt virus (SPCSV) are a severe problem in the production of sweetpotato (Ipomoea batatas L.). Traditional molecular virus detection methods include nucleic acid-based and serological tests. In this study, we aimed to validate the use of a non-destructive imaging-based plant phenotype platform to study plant-virus synergism in sweetpotato by comparing four virus treatments with two healthy controls. Results By monitoring physiological and morphological effects of viral infection in sweetpotato over 29 days, we quantified photosynthetic performance from chlorophyll fluorescence (ChlF) imaging and leaf thermography from thermal infrared (TIR) imaging among sweetpotatoes. Moreover, the differences among different treatments observed from ChlF and TIR imaging were related to virus accumulation and distribution in sweetpotato. These findings were further validated at the molecular level by related gene expression in both photosynthesis and carbon fixation pathways. Conclusion Our study validated for the first time the use of ChlF- and TIR-based imaging systems to distinguish the severity of virus diseases related to SPFMV and SPCSV in sweetpotato. In addition, we demonstrated that the operating efficiency of PSII and photochemical quenching were the most sensitive parameters for the quantification of virus effects compared with maximum quantum efficiency, non-photochemical quenching, and leaf temperature.Peer reviewe

Yield reduction caused by a soil-borne disease of naked, dwarf, and conventional oat in Finland

A severe disease occurred in the field plots of naked (cv. Salomon), dwarf (cv. Pal), and conventional oat (cvs. Jalostettu maatiainen and Salo) at the Viikki Experimental Farm of the University of Helsinki, Finland, in 1994 and 1995. Symptoms were expressed as grayish-brown necrotic areas on the lower leaves which killed plants from the seedling to heading stage, the effect being cultivar dependent. The proportion of plants killed contributed to the yield losses. The infection also resulted in less grains per panicle and lower weight of both panicle and vegetative above-ground biomass. From a total of 57 fungal isolates obtained from infected leaves, Fusarium culmorum (W.G.Sm.) Sacc. and F. sambucinum Fuck, dominated and subsequently caused infection (particularly foot and root rot) in oat in laboratory tests. These two Fusarium spp. were considered to be the primary causal agents of the symptoms observed in the field, although other pathogens may have been present. The disease was probably soil-borne. The results of this study suggested that the unusually dry and warm weather during late June and in July was the principal factor behind the severe disease outbreak

Small-RNA Deep Sequencing Reveals Arctium tomentosum as a Natural Host of Alstroemeria virus X and a New Putative Emaravirus

Peer reviewe

Nuclear proteome of virus-infected and healthy potato leaves

BackgroundInfection of plants by viruses interferes with expression and subcellular localization of plant proteins. Potyviruses comprise the largest and most economically damaging group of plant-infecting RNA viruses. In virus-infected cells, at least two potyviral proteins localize to nucleus but reasons remain partly unknown.ResultsIn this study, we examined changes in the nuclear proteome of leaf cells from a diploid potato line (Solanum tuberosum L.) after infection with potato virus A (PVA; genus Potyvirus; Potyviridae) and compared the data with that acquired for healthy leaves. Gel-free liquid chromatography-coupled to tandem mass spectrometry was used to identify 807 nuclear proteins in the potato line v2-108; of these proteins, 370 were detected in at least two samples of healthy leaves. A total of 313 proteins were common in at least two samples of healthy and PVA-infected leaves; of these proteins, 8 showed differential accumulation. Sixteen proteins were detected exclusively in the samples from PVA-infected leaves, whereas other 16 proteins were unique to healthy leaves. The protein Dnajc14 was only detected in healthy leaves, whereas different ribosomal proteins, ribosome-biogenesis proteins, and RNA splicing-related proteins were over-represented in the nuclei of PVA-infected leaves. Two virus-encoded proteins were identified in the samples of PVA-infected leaves.ConclusionsOur results show that PVA infection alters especially ribosomes and splicing-related proteins in the nucleus of potato leaves. The data increase our understanding of potyvirus infection and the role of nucleus in infection. To our knowledge, this is the first study of the nuclear proteome of potato leaves and one of the few studies of changes occurring in nuclear proteomes in response to plant virus infection.Peer reviewe