Rosette Virus Research Articles

Identification of a Second Vector for Rose Rosette Virus.

Rose rosette devastates the ornamentals industry in the United States. The disease, caused by rose rosette emaravirus (RRV), is vectored by the eriophyoid mite Phyllocoptes fructiphilus (Acari: Eriophyoidea). In this communication, we investigate two other Phyllocoptes species, P. adalius and P. arcani, for their vector competency and transmission efficiencies in single and multiple mite transfer experiments. P. arcani was identified as a second vector of RRV, a finding of significance for the epidemiology of the disease, as the second vector may be present in plants where P. fructiphilus is absent.

Genetic Diversity among Rose Rosette Virus Isolates: A Roadmap towards Studies of Gene Function and Pathogenicity.

The phylogenetic relationships of ninety-five rose rosette virus (RRV) isolates with full-length genomic sequences were analyzed. These isolates were recovered mostly from commercial roses that are vegetatively propagated rather than grown from seed. First, the genome segments were concatenated, and the maximum likelihood (ML) tree shows that the branches arrange independent of their geographic origination. There were six major groups of isolates, with 54 isolates in group 6 and distributed in two subgroups. An analysis of nucleotide diversity across the concatenated isolates showed lower genetic differences among RNAs encoding the core proteins required for encapsidation than the latter genome segments. Recombination breakpoints were identified near the junctions of several genome segments, suggesting that the genetic exchange of segments contributes to differences among isolates. The ML analysis of individual RNA segments revealed different relationship patterns among isolates, which supports the notion of genome reassortment. We tracked the branch positions of two newly sequenced isolates to highlight how genome segments relate to segments of other isolates. RNA6 has an interesting pattern of single-nucleotide mutations that appear to influence amino acid changes in the protein products derived from ORF6a and ORF6b. The P6a proteins were typically 61 residues, although three isolates encoded P6a proteins truncated to 29 residues, and four proteins extended 76-94 residues. Homologous P5 and P7 proteins appear to be evolving independently. These results suggest greater diversity among RRV isolates than previously recognized.

An artificial positive control for routine detection of rose rosette virus and Phyllocoptes fructiphilus that fit most primers for PCR, LAMP and RPA based assays

AbstractRose (Rosa spp.) is a very important ornamental shrub cultivated worldwide and of value for the pharmaceutical industry. The plant is broadly susceptible to pathogens, including viruses. Rose rosette virus (RRV; virus species Emaravirus rosae) causes multiple symptoms typically rosettes, ultimately leading to death. The virus transmission is by grafting and a wind‐dispersed eriophyoid mite, Phyllocoptes fructiphilus, which survives in winter‐dormant plants. Due to extensive globalization RRV is a threat for the European rose, landscape, nursery and tourism industries. The most common and reliable method used for RRV detection is RT‐PCR. Positive control is indispensable for PCR reliability and can be difficult to obtain for emerging or highly contagious pathogens and are subject to BSL‐2 quarantine. A synthetic artificial positive control (APC) using custom DNA inserts of sense and anti‐sense primers was designed de novo and inserted in a circular plasmid vector to create a positive control for use with most RRV reported primers and eriophyoid mites. This study describes a functional demonstration and development of a rapid, consistent, adaptable and cost‐effective alternative to infected true‐tissue positive control for detection of RRV. The inserted RRV primers are for end point and quantitative RT‐PCR, reverse transcription loop‐mediated isothermal amplification (RT‐LAMP), recombinase‐polymerase amplification (RPA), broad detection of emaravirus and the eriophyoid mite vector Phyllocoptes fructiphilus. The APC‐RRV and RRV infected rose (leaf tissue) were tested side to side. Results demonstrated APC‐RRV is a safe, cloneable and reliable approach subjected to quality control with application in quarantine surveillance and routine diagnostics of RRV.

Exploring the Host Range of Rose rosette Virus among Herbaceous Annual Plants.

To study the host range of Rose rosette virus (RRV), we employed crude sap inoculum extracted from RRV-infected roses and the RRV infectious clone. We inoculated plants from the families Solanaceae, Cucurbitaceae, Leguminosae, Malvaceae, Amaranthaceae, and Brassicaceae. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect RRV in the inoculated plants throughout their growth stages. Interestingly, RRV was detected in the newly developed leaves of tomato, pepper, tobacco, cucumber, squash, zucchini, pumpkin, pea, peanut, soybean, spinach, okra, and Chenopodium spp. The speed of upward advancement of RRV within infected plants was variable between plants as it took two to three weeks for some plant species and up to five weeks in other plant species to emerge in the newest leaves. No severe symptoms were detected on most of the inoculated plants. Chenopodium spp., spinach, cucumber and Nicotiana rustica exhibited either chlorotic or necrotic lesions with variable shapes and patterns on the systemically infected leaves. Double membrane-bound particles of 80-120 nm in diameter were detected by transmission electron microscopy in the infected tissues of cucumber, pepper, and N. benthamiana plants. This finding infers the validity of mechanical inoculation for RRV on a wide range of plants that would serve as potential natural reservoirs.

Phenology and effects of relative humidity and temperature on Phyllocoptes fructiphilus*

Rose rosette disease (RRD) is a serious rose disease in North America. RRD is caused by rose rosette virus transmitted by Phyllocoptes fructiphilus (Acari: Eriophyidae).

Pseudomonas aeruginosa isolate PM1 effectively controls virus infection and promotes growth in plants.

A bacterial isolate PM1 obtained from the rhizosphere of healthy plants was identified as Pseudomonas aeruginosa by biochemical characteristics and 16S rRNA gene sequence (GenBank ID OL321133.1). It induced resistance in Nicotiana tabacum cv. Xanthi-nc and Cyamopsis tetragonoloba, against Tobacco mosaic virus (TMV) and Sunn-hemp rosette virus (SRV), respectively. Foliar treatment with isolate PM1 curbed TMV accumulation in susceptible N. tabacum cv. White Burley. PM1 was more effective as a foliar than a root/soil drench treatment, evident through a comparative decrease in ELISA values, and reduced viral RNA accumulation. Foliar and soil drench treatment with PM1 resulted in a disease index of 48 and 86 per cent, and a control rate of 48.9 and 8.5 per cent, respectively. PM1 exhibited phosphate solubilization, produced siderophores, auxins, HCN, and ammonia, all important plant growth-promoting traits. Foliar treatment with PM1 enhanced growth in tobacco, while its volatiles significantly promoted seedling growth in C. tetragonoloba. Of the several metabolites produced by the isolate, many are known contributors to induction of systemic resistance, antibiosis, and growth promotion in plants. Soluble metabolites of PM1 were less effective in inducing antiviral resistance in N. tabacum cv. Xanthi-nc in comparison with its broth culture. PM1 and its metabolites were antagonistic to Gram-positive Bacillus spizizenii and Staphylococcus aureus, and fungi Fusarium oxysporum, Aspergillus niger, and Rhizopus stolonifer. Its volatiles were inhibitory to F. oxysporum and R. stolonifer. Thus, PM1 exhibited considerable potential for further evaluation in plant virus control and production of diverse metabolites of use in agriculture and medicine.

Exploration of Alternative Approaches to Phenotyping of Late Leaf Spot and Groundnut Rosette Virus Disease for Groundnut Breeding

Late leaf spot (LLS), caused by Nothopassalora personata (Berk. & M.A Curt.), and groundnut rosette disease (GRD), [caused by groundnut rosette virus (GRV)], represent the most important biotic constraints to groundnut production in Uganda. Application of visual scores in selection for disease resistance presents a challenge especially when breeding experiments are large because it is resource-intensive, subjective, and error-prone. High-throughput phenotyping (HTP) can alleviate these constraints. The objective of this study is to determine if HTP derived indices can replace visual scores in a groundnut breeding program in Uganda. Fifty genotypes were planted under rain-fed conditions at two locations, Nakabango (GRD hotspot) and NaSARRI (LLS hotspot). Three handheld sensors (RGB camera, GreenSeeker, and Thermal camera) were used to collect HTP data on the dates visual scores were taken. Pearson correlation was made between the indices and visual scores, and logistic models for predicting visual scores were developed. Normalized difference vegetation index (NDVI) (r = –0.89) and red-green-blue (RGB) color space indices CSI (r = 0.76), v* (r = –0.80), and b* (r = –0.75) were highly correlated with LLS visual scores. NDVI (r = –0.72), v* (r = –0.71), b* (r = –0.64), and GA (r = –0.67) were best related to the GRD visual symptoms. Heritability estimates indicated NDVI, green area (GA), greener area (GGA), a*, and hue angle having the highest heritability (H2 > 0.75). Logistic models developed using these indices were 68% accurate for LLS and 45% accurate for GRD. The accuracy of the models improved to 91 and 84% when the nearest score method was used for LLS and GRD, respectively. Results presented in this study indicated that use of handheld remote sensing tools can improve screening for GRD and LLS resistance, and the best associated indices can be used for indirect selection for resistance and improve genetic gain in groundnut breeding.

Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor.

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.

Temporal Incidence of Eriophyid Mites on Rose Rosette Disease-Symptomatic and -Asymptomatic Roses in Central Georgia, USA.

Simple SummaryRose rosette disease (RRD) is a serious disease of rose caused by the rose rosette virus (RRV). An eriophyid mite, Phyllocoptes fructiphilus, is the vector of RRV. The RRD symptoms affect the normal growth and development of rose plants. Because there is no cure for RRD, this disease threatens the rose industry, including container nurseries and cut flowers in the U.S. The seasonal occurrence and abundance of the vector and the locations they colonize on the plant are poorly studied in Georgia. The eriophyid mites are active from April to December on rose plants. The eriophyid mites were more abundant on the plants with RRD symptoms than on plants without any symptoms. The mites were found on both closed and opened flower buds alike. More mites were found on leaf bases and sepals than on other plant parts, such as leaf surfaces, stem, petals, anthers, stigma, and style. These results will help to develop integrated pest management strategies for the mite vector and reduce the spread of RRD.Phyllocoptes fructiphilus Keifer (Acari: Eriophyidae) is the vector of rose rosette virus (RRV), which causes rose rosette disease (RRD) in North America. The RRD symptoms, such as witches’ broom, flower, and leaf deformation, disrupt the aesthetic appearance of plants and cause plant mortality. Because there is no cure for RRV, it is critical to manage the vector and reduce the spread of the virus. The information on the phenology of P. fructiphilus on rose plants is essential to develop management strategies and reduce its spread. Thus, the objectives of the study were to determine 1) the phenology of eriophyid mites (including P. fructiphilus) in central Georgia due to its widespread occurrence in the state and 2) the incidence of eriophyid mites on closed and opened flower buds and other plant parts. In central Georgia, eriophyid mites, including P. fructiphilus were active on both symptomatic and asymptomatic plants from April to December. The mite densities were greater during July and August than during the remaining months on asymptomatic plants. The mites were more abundant on the RRD-symptomatic than on the asymptomatic plants. Similar numbers of eriophyid mites were observed on closed and opened flower buds. Eriophyid mite densities were greater on sepals and leaf bases than on other plant parts.

Rose Rosette Disease: A Diagnostic Guide

Rose rosette disease (RRD) is a common disease of roses, predominantly in North America. RRD is caused by the rose rosette virus (RRV), an emaravirus transmitted by the eriophyid mite Phyllocoptes fructiphilus Keifer. RRD characteristic symptoms include deformed leaves, abnormal thorns, and witches’ brooms. Roses are important cultivated plants around the world. Most Rosa spp. and cultivated varieties such as climbers, hybrid teas, floribundas, and miniature roses are susceptible to the disease. Rose rosette virus detection relies on molecular assays.

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.

Molecular phylogeny of Phyllocoptes associated with roses discloses the presence of a new species.

There are few plant maladies as devastating as rose rosette, a disease caused by an eriophyoid -transmitted virus. Rosette annihilates roses across North America, and to date, there is a single verified vector of the virus, Phyllocoptes fructiphilus Keifer. In direct contrast to the importance of rose for the ornamental industry there is limited knowledge on the eriophyoids that inhabit roses in North America and even less information on their vectoring capacities. This study dissects the genetic diversity of the eriophyoid fauna in rosette-affected hotspots and provides evidence of the existence of an undescribed species named Phyllocoptes arcani sp. nov., that could potentially be a second vector of the rosette virus.

Effects of Relative Humidity on the Vector of Rose Rosette Disease, Phyllocoptes fructiphilus (Eriophyidae), and Incidence of Disease Symptoms

The eriophyid mite Phyllocoptes fructiphilus Keifer (Acari: Eriophyidae) transmits rose rosette virus to rose (Rosa spp.; Rosaceae) while feeding, which causes serious disease referred to as rose rosette disease. Although there is no cure once rose plants are infected with rose rosette virus, understanding the response of P. fructiphilus and rose rosette disease to abiotic factors such as relative humidity may help to develop management strategies for the disease. The major objective of the current study was to determine the effect of relative humidity on the abundance of P. fructiphilus, as well as the incidence and severity of rose rosette disease symptoms. An experiment was conducted in environmentally controlled chambers where potted pink double knock-out rose plants were maintained at 20, 60, and 95% relative humidity after introducing about 20 P. fructiphilus individuals by attaching a 7-cm-long, field-collected terminal to the branches of potted plants. The densities of P. fructiphilus were recorded at biweekly intervals for 12 wk. The proportion of terminals with rose rosette disease symptoms (disease incidence) and severity of rose rosette disease symptoms was assessed using the Horsfall-Barratt scale at biweekly intervals for 14 wk. The results show that the number of P. fructiphilus individuals was significantly greater under a moderate 60% relative humidity than under a high (95%) or low (20%) relative humidity (P < 0.05). However, the incidence and severity of rose rosette disease symptoms were significantly higher under 95% relative humidity relative humidity regimen than under 20% relative humidity regimen (P < 0.05). The implications of these results on the breeding program and management of P. fructiphilus and the incidence of rose rosette disease are discussed.

First report of Rose leaf rosette-associated virus infecting rose (Rosa spp.) in California, USA.

Rose leaf rosette-associated virus (RLRaV) is a member of genus Closterovirus, family Closteroviridae. The virus was first discovered in China in 2015 from a mixed infected wild rose (Rosa multiflora Thunb.) showing small leaf rosettes on branches, dieback and severe decline symptoms (He et al. 2015). In 2013, a rose plant (cv. Roses Are Red) was introduced to Foundation Plant Services (FPS, UC-Davis) rose collection. The plant was originated from a private rose breeder collection located in California. In 2019, total nucleic acids (TNA) were isolated from leaf tissues of one asymptomatic plant (Roses Are Red plant) using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific, USA). Extracted TNA were screened by reverse-transcription quantitative PCR (RT-qPCR) for six common viruses infecting roses, including prunus necrotic ringspot virus (PNRSV), apple mosaic virus (ApMV), rose spring dwarf associated virus (RSDaV), rose yellow vein virus (RYVV), rose rosette virus (RRV), and blackberry chlorotic ringspot virus (BCRV); however, the results were negative. Therefore, the sample was subjected to high throughput sequencing (HTS). Briefly, TNA was depleted of rRNA and advanced for cDNA library preparation using TruSeq Stranded Total RNA kit (Illumina, USA). HTS was performed on Illumina NextSeq 500 platform. The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). HTS generated 23.6 million 75 nucleotide (nt) single-end raw data reads. De novo assembly generated a contig (16,528 nts) resembling RLRaV reference sequence (KJ748003) with 74% identity at the nucleotide level. Putative coat protein and heat shock protein 70-like protein were identified based on >90% identity with RLRaV genes. To confirm HTS results, RT-PCR was performed using two primer sets, 1) Clo-F4916 (5'-GGTGTTCCAACGCTATCGTG-3') and Clo-R5215 (5'- TGTCCTCAAACCGCCTACAT-3') targeting nucleotide sequences of putative polyprotein 1a, and 2) Clo-F10006 (5'-GATTCCGCGGACGAATTAAT-3') and Clo-R10311 (5'-GGTAACCGAAAGGTAAAGTATTC-3') targeting nucleotide sequences of putative protein p25. The RLRaV amplicons with expected size of 300 nt were confirmed using bidirectional Sanger sequencing. The near-complete sequence of the new RLRaV isolate was deposited in GenBank under accession number MW056181. In addition, HTS analysis showed that RLRaV was in mixed infection with two mycoviruses (rose cryptic virus with 8,267 mapped reads and rose partitivirus with 7,283 mapped readss). To our knowledge, this is the first report of RLRaV affecting roses in California. Further research is needed to determine the prevalence of RLRaV in California as well as evaluation of RLRaV effect on rose performance.

Prevalence and molecular characterization of Turkish isolates of the rose viruses

Roses are among the most important ornamental plants in the world. There are many biotic disease factors that cause damage to this important plant. The most important group among these is virus diseases. However, of the studies about rose viruses conducted worldwide, those in Turkey are limited. With the aim of determining the prevalence and genetic diversity of rose viruses, 332 rose plants showing virus and virus-like symptoms were collected in 14 provinces from four different geographic regions of Turkey. The samples were tested to determine the infection of nine different viruses infecting rose plants. As a result of the tests, prunus necrotic ringspot (PNRSV), rose cryptic virus-1 (RCV-1), rose spring dwarf-associated virus (RSDaV), rose yellow vein virus (RYVV), and apple mosaic virus (ApMV) infections were detected. It was determined that 94.27% of the samples were infected with at least one virus infection. While the virus causing the most infection was found to be PNRSV, the least one was determined as ApMV. With this result, PNRSV is determined as a significant virus disease for rose plants in Turkey. Based on their geographical origins, 25 PNRSV and RCV-1 isolates, 15 RSDaV and RYVV isolates, and 10 ApMV isolates were selected for further analysis and their partial genetic diversity was investigated. As a result of these analyzes, it was determined that PNRSV, RCV-1, and RSDaV isolates showed a high-sequence homology with each other and with world isolates. Rose ApMV isolates were found to form separate groups phylogenetically. RYVV, which is known to exist in a very limited number of countries, has been found to have up to 10% genetic differences and phylogenetically, three different groups were determined. Furthermore, to the best of our knowledge, these are the first reports of RCV-1 and RSDaV in Turkey. It was also found that Turkey is still free of rose rosette virus. Therefore, quarantine measurements should continue to be strictly enforced.

Next-Generation Sequencing Reveals a Novel Emaravirus in Diseased Maple Trees From a German Urban Forest.

While the focus of plant virology has been mainly on horticultural and field crops as well as fruit trees, little information is available on viruses that infect forest trees. Utilization of next-generation sequencing (NGS) methodologies has revealed a significant number of viruses in forest trees and urban parks. In the present study, the full-length genome of a novel Emaravirus has been identified and characterized from sycamore maple (Acer pseudoplatanus) – a tree species of significant importance in urban and forest areas – showing leaf mottle symptoms. RNA-Seq was performed on the Illumina HiSeq2500 system using RNA preparations from a symptomatic and a symptomless maple tree. The sequence assembly and analysis revealed the presence of six genomic RNA segments in the symptomatic sample (RNA1: 7,074 nt-long encoding the viral replicase; RNA2: 2,289 nt-long encoding the glycoprotein precursor; RNA3: 1,525 nt-long encoding the nucleocapsid protein; RNA4: 1,533 nt-long encoding the putative movement protein; RNA5: 1,825 nt-long encoding a hypothetical protein P5; RNA6: 1,179 nt-long encoding a hypothetical protein P6). Two independent NGS sequencing runs from the same symptomatic maple tree detected the same genome segments. For one of these sequencing runs the cDNA library was prepared using a primer targeting the conserved genome terminal region, known to be shared between emaraviruses genome segments. We suggest, therefore, that the six identified genome segments represent the complete genome of a novel emaravirus from maple, which we tentatively name maple mottle-associated virus (MaMaV). Phylogenetic and sequence homology analyses place this virus on the distinct “subgroup a” clade within the Emaravirus genus along with – among others – rose rosette virus, Actinidia emaravirus 2, and fig mosaic virus. Validation RT-PCR assays performed on symptomatic and non-symptomatic trees suggest that MaMaV may be the symptom-inducing virus in the diseased trees. To our knowledge, this is the first time an Emaravirus is described from maple and is fully genetically characterized. With the discovery of MaMaV, the genus Emaravirus comprising negative-sense single-stranded viruses with very divergent genomes – that were until recently overlooked – has substantially increased counting 22 established and putative members.

Improved Groundnut Performance under Natural Field Leaf Spot and Rosette Mosaic Virus Infections in the South Western Agro-Ecological Zone of Uganda

For farmers to satisfy the food, nutrition and income demands on groundnut in the South Western Agro-Ecological Zone (SWAEZ), the accessibility, and adaptability of economically viable and high performing varietal and non-varietal technologies must increase to enhance production. Field experiments to evaluate the agronomic performance of advanced lines of improved nine AGRA red, nine Spanish and six Serenut varieties were concurrently carried out on-station at the Mbarara Zonal Agricultural Research and Development Institute (MbaZARDI) in Mbarara District, Uganda. The AGRA red and Spanish groundnut lines showed high levels of rosette mosaic disease (GRD) tolerance in the zone. The short AGRA and Spanish lines were highly resistant to leaf spots, while the tall lines were highly susceptible to leaf spots, and not drought tolerant. The late maturing AGRA lines (SGV 99241 and SGV 99046), yielded better than the early maturing lines (SGV 99065 and SGV 99043). The best performing AGRA lines identified for promotion in the zone were SGV 99241, 99046, 99064, 99048, 99019 and 99032. The most leaf spot-resistant Spanish line was ICGV SM 02501, but lines ICGV SM 01514, ICGV SM 03590, ICGV SM 01515 and ICGV SM 01502 displayed varying levels of resistance during the two seasons of 2015. Spanish lines: ICGV SM 01502, ICGV SM 99568, ICGV SM 01504, ICGV SM 01510, ICGV SM 99555, ICGV SM 01514 and ICGV SM 01515 were suitable for growing in the SWAEZ. Spanish lines that were either highly resistant or resistant to groundnut rosette virus disease (GRD), unfortunately, were either moderately-susceptible or susceptible to the leaf spot diseases. Therefore, the tested Spanish lines were only suitable to environments that are prone to GRD but free of the leaf spot pathogens. AGRA red lines SGV 99241, SGV 99046, SGV 99064, SGV 99048, SGV 99019 and SGV 99032 were the best performing. Of the Serenut varieties evaluated, Serenut 5R was early maturing and rosette mosaic virus tolerant. Apart from Serenut 1; Serenut 2, 3R, 4R, 5R and 6T displayed very high levels of tolerance to rosette mosaic virus disease. The combination of moderate resistance (MR)-resistance (R) and high resistance (HR) in the AGRA red lines and Serenut varieties to leaf spots and rosette mosaic virus, respectively, indicated an effective control measure of the major groundnut disease problems in the zone. The integration of moderate-resistance to high disease resistance, high drought tolerance and high yielding potential in several Serenut, AGRA red and Spanish lines can enhance groundnut production if proper agronomic management practices and timely planting are applied.

Next generation sequencing as a tool in modern pest risk analysis: a case study of groundnuts (Arachis hypogaea) as a potential host of new viruses in western Kenya

Groundnut (Arachis hypogaea, L.) is grown in diverse environments throughout the semi-arid and sub-tropical regions of the world. Poor yields of 500-800kg/ha are attributed to poor agronomic practices, pests and diseases. The major disease reported in Kenya is Groundnut rosette disease (GRD). But recent observations in the field showed that the crop has varied and severe symptoms in addition to those caused by GRD. This required deeper analysis to establish the causal agents. Groundnut samples with virus-like symptoms were collected from western Kenya in 2016. Total RNA was extracted using All Prep RNA Mini Kit. Five mRNA libraries were prepared using the Illumina TrueSeq stranded mRNA library Prep Kit and pooled for multiplexed sequencing using an Illumina HiSeq 2500 to generate paired end reads (FastQ Sanger). The reads were analysed in the Galaxy project platform (customized). Quality reads were first mapped onto plant genome Refseq and unmapped reads isolated and mapped onto virus Refseq using Bowtie 2 (v2.2.3). Groundnut rosette virus satellite RNA, Groundnut rosette virus, Groundnut rosette assistor virus, Ethiopian tobacco bushy top virus, Cowpea polerovirus 2, Chickpea chlorotic stunt virus, Melon aphid-borne yellow virus, Phasey bean mild yellow virus, Beet mild yellowing virus, White clover mottle virus and Cotton leafroll dwarf virus were identified in four libraries. Other viruses (with less than 100 reads) including Bean common mosaic virus, Bean common mosaic necrosis virus, Cowpea chlorotic mottle virus RNA 3, Broad bean mottle virus RNA 3, Passion fruit woodiness virus among others were also mapped. Some of the viruses common in western Kenya were confirmed by PCR. The presence of at least three viruses in groundnuts in Western Kenya highlights the importance of starting a germplasm clean-up program of the plant material used as seed in this crop. Key words: Groundnuts, NGS, RefSeq, Viruses.

First Report of Phyllocoptes fructiphilus Keifer (Eriophyidae), the Vector of the Rose Rosette Virus, in Florida, USA

The invasive mite Phyllocoptes fructiphilus Keifer (Acari: Eriophyidae) feeds on plants in the genus Rosa. Phyllocoptes fructiphilus is associated with the rose rosette emaravirus (rose rosette virus) and acts as the only known vector of rose rosette virus, the causal agent of rose rosette disease (Emaravirus). The mite P. fructiphilus is reported for the first time in the state of Florida, USA. No roses showed signs or symptoms of viral infection, and current molecular methods were unable to detect the virus. Phyllocoptes fructiphilus represents a potential threat to the Florida rose industry if rose rosette disease becomes established.

Occurrence and genetic diversity of groundnut rosette assistor virus in western Kenya

Groundnut (Arachis hypogaea) is an important legume in western Kenya, but yields are low and declining. Pests and diseases are ranked high among the yield reducing factors. Groundnut rosette disease (GRD) is the main disease and can cause up to 100% yield loss. Groundnut rosette disease is transmitted by the groundnut aphid, Aphis craccivora Koch and caused by two synergistic viruses; groundnut rosette assistor virus (GRAV) and groundnut rosette virus (GRV) associated with a satellite-ribonucleic acid (sat-RNA). Inadequate information was available on the occurrence and molecular diversity of GRAV in western Kenya, which is a limiting factor on proper diagnosis and management. This study determined the occurrence and molecular diversity of GRAV on groundnuts in western Kenya. A survey of GRD was conducted in 144 farms in Bungoma and Kakamega counties during the short and long rains of 2016–2017. A total of 301 symptomatic leaf samples were collected and preserved until use. Incidence and severity data was collected and subjected to pairwise comparison of means using the standardized disease diagnostic score sheet at P ≤ 0.05. Screening for resistance/tolerance to GRD was done on five popular legume varieties (Arachis hypogaea, Phaseolus vulgaris, Vigna unquiculata, Glycine max and Vigna radiata) and one solanaceous Physalis peruviana. The plants were monitored for GRD symptoms development in the screenhouse for 8 weeks. Total RNA was extracted from the leaf samples using RNeasy Mini Kit (Qiagen) according to the manufacturers’ protocol. DNA libraries were prepared and sequenced on the MiSeq platform (Illumina). Phylogenetic analyses and comparisons were performed using the MEGA X software. From the sequences obtained, primers were designed for use in RT-PCR. Groundnut rosette disease was prevalent in all the surveyed areas with chlorotic rosette symptoms being dominant followed by green rosette and mosaic rosette. Groundnut rosette disease incidence ranged between 31 and 67%, but was higher in Bungoma (67%) than Kakamega (61%) county, while the short rains had higher incidence than the long rains season. All screened indicator plants developed typical GRD viral symptoms and tested positive for GRAV. Nucleotide sequences of GRAV coat protein (CP) gene revealed 97–99% identity among the western Kenya isolates and those from Ghana, Malawi and Nigeria respectively. The fact that GRD occurs wherever groundnuts are grown in western Kenya, is of great concern and may be the reason for the observed low yields. Incorporation of GRD resistance genes in the local cultivars/varieties may be the only practical solution.