Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus

This study explores the development of Loop-mediated isothermal amplification (LAMP) for detection of rose rosette virus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense, single-stranded RNA virus which is a member of the genus Emaravirus (Family Fimoviridae) and the causal agent of the rose rosette disease (RRD). Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear like herbicide damage. Moreover, it may take incubation time for symptoms to appear after virus infection. Two sets of RRV gene sequences RNA3 and RNA4 were analyzed and two sets of four LAMP primers were designed. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. RT-LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. RT-qLAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) was 1pg/μL and 1 fg/μL using Bst 2.0 LAMP and GspSSD LD quantitative LAMP, respectively. In visual colorimetric pre- and post-reactions, the LoD was 10 pg/μL and 0.1 pg/μL using hydroxy naphthol blue (HNB, 120 μM) and SYBR green I (1:10 dilution), respectively. No cross-reactivity was detected in the RT-LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses and one virus taxonomically related to RRV. Four different dyes were tested, and visible colorimetric reactions were obtained with RT-LAMP Bst 2.0 combined with SYBR I or HNB. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR and it is faster since it works with RNA directly.

Impact of Foliar Application of Acibenzolar S-Methyl on Rose Rosette Disease and Rose Plant Quality

Rose rosette disease (RRD) caused by rose rosette emaravirus (RRV) is a major issue in the U.S. rose industry with no effective method for its management. This study evaluated the effect of foliar application of Acibenzolar S-methyl (ASM), a plant systemic acquired resistance inducer in reducing RRD disease severity on Rosa species cv. Radtkopink (Pink Double Knock Out®) under greenhouse condition, and the effect of ASM on plant growth under commercial nursery production conditions. ASM at 50 or 100 mg/L at weekly intervals significantly reduced RRD severity compared to the untreated control in two of the three greenhouse trials (P < 0.05). The plants in these trials were subsequently pruned and observed for symptoms, which further indicated that application of ASM at 50 or 100 mg/L lowered disease severity compared to the untreated control (P < 0.05) in these two trials. Plants treated with ASM at 50 or 100 mg/L had delayed incidence of RRD compared to the non-treated controls. Plants treated with ASM at 50 or 100 mg/L rate in all three trials either did not have RRV present or the virus was present in fewer leaf samples than untreated controls as indicated by RT-qPCR analysis. Overall, plants treated with ASM at 50 mg/L had 36-43% reduced RRD incidence compared to the water control. The treatment of two cultivars of rose, ‘Radtkopink’ and ‘Meijocos’ (Pink Drift®), with weekly foliar applications of ASM at three rates (0.5, 0.75 and 1.0 oz/A) indicated that ASM had no negative effect on flowering or plant growth at even the highest rate.

When the Trauma Draws a Rose: Rosette Cataract

A 40-year-old male patient referred to the ophthalmologic emergency room for complaints of diminution of vision in the left eye since three months after a trauma. Examination of the left eye showed a corrected visual acuity of 20/70. Slit-lamp examination revealed a rosette cataract (Figure 1). The patient underwent phacoemulsification with implantation of an IOL in the bag. The rosette cataract is a posterior subcapsular cataract due to a violent contusion with a closed globe. It can develop in the hours that follow, or on the contrary several years later.

Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus

This study explores the development of Loop-mediated isothermal amplification of DNA (LAMP) for detection of rose rosette emaravirus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense single-stranded RNA Emaravirus and causal agent of the rose rosette disease (RRD). Transmission of RRV is by Phyllocoptes fructiphilus, an eriophyid mite. Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear similar to herbicide damage. Two sets of RRV gene sequences composed of twenty-two accessions of RRV-P3 (RNA 3) and another twenty-four from RRV-P4 (RNA 4) were analyzed and two sets of four LAMP primers were designed for broad-range detection of RRV isolates. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. LAMP reactions were optimized for Bst 2.0 DNA polymerase using the outer RRV-F3/RRV-B3 primers, and internal RRV-FIP/RRV-BIP primers. LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. LAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) using Bst 2.0 LAMP was 1pg/μL and 1 fg/μL with GspSSD LD quantitative LAMP. The LoD of pre-reaction hydroxy naphthol blue (HNB, 120 μM) for colorimetric (visual) reactions was 10 pg/μL and 0.1 pg/μL using SYBR green I (1:10 dilution) in colorimetric post-reactions. No cross-reactivity was detected in LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses (INSV, ArMV, MSpV, TSWV, ApMV, PNRSV, ToRSV, and TMV), and one virus taxonomically related to RRV (HPWMoV). RNA from healthy rose tissues and non-template controls (water) were included in all LAMP assays.

Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants

Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

A Survey of Rose rosette virus and Eriophyid Mites Associated with Roses in the Southeastern United States

The eriophyid mite, Phyllocoptes fructiphilus, vectors the causal agent, Rose rosette virus (RRV), that results in rose rosette disease. Parts of the southeastern United States have remained free of the disease, except for infected plant material introductions that were eradicated. A survey of sampling points through Alabama, Georgia, and Mississippi (n = 204) revealed the southeastern border of RRV. The presence of RRV in symptomatic plant tissue samples (n = 39) was confirmed by TaqMan-quantitative reverse transcription polymerase chain reaction (RT-qPCR). Samples were also collected at every plot for detection of eriophyid mites, specifically for P. fructiphilus. Three different species of eriophyid mites were found to be generally distributed throughout Alabama, Georgia, and Mississippi. Most of these sites (n = 60) contained P. fructiphilus, found further south than previously thought, but in low populations (<10 mites/gram of tissue) south of the RRV line of incidence. Latitude was found to be significantly correlated with the probability of detecting RRV-positive plants, but plant hardiness zones were not. Plot factors such as plant size, wind barriers, and sun exposure were found to have no effect on P. fructiphilus or the presence of RRV. The reason for the absence of RRV and low populations of P. fructiphilus in this southeast region of the United States are unclear.