Chlorotic Rosette Research Articles

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.

Distribution of groundnut rosette disease and sequence diversity of groundnut rosette virus associated satellite RNA (Sat-RNA) in Western Kenya

Production of groundnuts (Arachis hypogaea L.) in Western Kenya is mainly constrained by groundnut rosette disease (GRD) which cause up to 100% yield loss. This disease expresses different symptoms as a result of variations in the groundnut rosette virus (GRV) associated satellite-ribonucleic acid (GRV Sat-RNA). Over the past 20 years, no work had been done to document the status of the disease in Kenya. Additionally, no sequences of any of the GRD associated viruses were available in the GeneBank from Kenya. This study determined the distribution of GRD and the genetic diversity of GRV Sat-RNA. Sampling was done in main groundnut growing areas of Western Kenya during the long and short rain seasons in 2016/2017. Total RNA was extracted from the leafy samples collected using RNeasy Mini Kit (Qiagen) according to the manufacturers’ protocol and used for double stranded cDNA synthesis using the SuperScript II kit. DNA libraries were sequenced on the Illumina MiSeq platform (Illumina). Reads were used for de novo assembly and contigs aligned to the viral genomes database using CLC Genomics Workbench 10.1.2. The assembled contigs were subjected to a BLASTn search against the GenBank database. Average GRD incidence was 53 and 41% in the short and long rain seasons, respectively. Chlorotic rosette was the dominant symptom followed by green rosette and mosaic. Nucleotide sequences of Sat-RNA revealed identities of 88 to 100% between the Kenyan isolates and those from Malawi, Nigeria and Ghana. All Kenya isolates clustered closest with green rosette variants of Malawi except one which clustered with chlorotic/yellow blotch variants. Rosette is widely distributed in Western Kenya and occurs wherever groundnuts are grown. The variations of GRD symptoms in Western Kenya could be due to the existence of different variants of Sat-RNA or other agents. Key words: Groundnuts, satellite-ribonucleic acid (Sat-RNA), diversity, Western Kenya.

Incidence of Groundnut Rosette Disease (GRD) and Genetic Diversity of Groundnut Rosette Assistor Virus (GRAV) in Western Kenya

This study determined the incidence of groundnut rosette disease (GRD) and genetic diversity of groundnut rosette assistor virus (GRAV, genus Luteovirus) in western Kenya. The diseases is a major constraint of groundnuts in Sub-Saharan Africa (SSA) causing up to 100% yield losses in severe cases. Among the GRD associated viruses, GRAV plays a crucial role in vector transmission of the other viruses. Therefore understanding the genetics of GRAV across SSA could enhance development of resistance to the disease. In Kenya, groundnuts are mainly grown in western region, however, the yields are poor mainly due to GRD. Information on occurrence and distribution of GRD in western Kenya was not documented and little was known about the characteristics of associated viruses. Two diagnostic surveys were conducted in six counties; Bungoma, Busia, Homabay, Kakamega, Siaya and Vihiga. Symptomatic and asymptomatic groundnut were collected in RNAlater® solution for laboratory analysis. Total RNA was extracted from the leaf samples using RNeasy Mini Kit (Qiagen) according to the manufacturers’ protocol and used for double stranded cDNA synthesis using the SuperScript II kit. The cDNA was column-purified with the DNA Clean & ConcentratorTM-5 – DNA kit. The samples were then processed with the transposon-based chemistry library preparation kit (Nextera XT, Illumina) following manufacturer’s instructions. The fragment sizes structure of the DNA libraries was assessed using the Agilent 2100 Bioanalyzer. The indexed denatured DNA libraries were sequenced (200-bp paired-end sequencing) on the Illumina MiSeq platform (Illumina). Reads quality check was done using FastQC. Trimmed reads were used for de novo assembly and contigs aligned to the viral genomes database using CLC Genomics Workbench 10.1.2. The assembled contigs were subjected to a BLASTn search against the GenBank database. Phylogenetic analyses and comparisons were performed using the MEGA X. Average incidence was 53% and 41% in the short and long rain seasons, respectively. Chlorotic rosette was the dominant symptom followed by Green rosette and Mosaic. The GRAV coat protein (GRAV-CP) gene sequences revealed 97-100% identity with GeneBank isolates showing very slight variations across SSA. The study concludes that GRD incidence is high in western Kenya and that GRAV is highly conserved across SSA. The study recommends an urgent need to curb GRD, possibly through the exploitation of pathogen derived resistance (PDR) with GRAV as the suitable candidate.

Molecular diagnostics of groundnut rosette disease agents in Uganda: Implications on epidemiology and management of groundnut rosette disease

The objective of this study was to use molecular diagnostic tools to detect the agents of groundnut rosette disease (GRD) to guide in varietal development and disease management. Samples were collected from both GRD infected and healthy plants and sites geo-referenced. RNA extraction, cDNA synthesis, polymerase chain reaction (PCR) amplification, electrophoresis, staining and visualization were performed according to standard procedures. Molecular diagnosis of the samples showed various combinations of the GRD agents, some in isolation and others a combination of two or three agents. This distribution is attributed to dependence on the aphid feeding behaviour and pathogenicity of GRD agents. Chlorotic and green rosette symptoms were observed throughout the sampling sites signifying the presence of satellite RNA (sat-RNA) variants. Some plants showing GRD symptoms tested negative for GRD, whereas some healthy-looking plants tested positive for the GRD complexes pointing to the ineffectiveness of phenotypic screening and the need for a molecular diagnostic tool that detects all three GRD agents both in absence or presence of disease symptoms. The absence of groundnut rosette assistor virus (GRAV) in some symptomatic samples signifies that they are epidemiologically dead end sources since GRV and sat-RNA must be packaged within the GRAV coat protein to be aphid transmissible. Oyado (Cassia obtusifolia) tested positive for all the GRD agents making it a potential alternative host. There is an urgent need for validation of the phenotypic screening with molecular tools in efficient diagnosis of the multi-pathogenic GRD in guiding both plant breeding and pathology work. Key words: Arachis hypogaea L., groundnut rosette disease, molecular diagnostics, polymerase chain reaction (PCR).

English

Groundnut chlorotic rosette disease (GCRD) transmitted by the aphid, Aphis craccivora, is an important virus disease of groundnut in Africa. Breeding for host resistance remains the best strategy to minimize losses due to this disease. Nine cultivated groundnut genotypes with differential reaction to GCRD were crossed in an incomplete diallel mating design to determine the combining ability of GCRD resistance. The parents and 36 F2 populations were inoculated with veruliferous A. craccivora at the seedling stage and evaluated for disease reaction at two locations in Nigeria in 2012. Disease incidence (based on visual symptoms) was recorded three times at fortnightly interval using area under disease progress curve. General combining ability (GCA) and specific combining ability (SCA) effects for GCRD resistance were highly significant (P < 0.01), indicating that both additive and non-additive gene effects governed the inheritance of GCRD resistance. The Baker ratio was low (0.3) for GCRD indicating that non-additive gene effects was more important than additive gene effects in controlling GCRD resistance in these crosses. As a result, progeny performance could not be adequately predicted from GCA effects alone. Therefore, effective selection of superior genotypes would be achieved at advanced generations when maximum homozygosity is attained. Key words: Groundnut chlorotic rosette disease, area under disease progress curve, combining ability, additive and non-additive gene effects.

English

Groundnut rosette disease (GRD), caused by a complex of three agents: groundnut rosette assistor luteovirus, groundnut rosette umbravirus, and the associated satellite RNA, is a major groundnut disease in Uganda. Two main symptom types, chlorotic rosette and green rosette occur. A nationwide survey covering 23 districts was done in 2012 and 2013 to ascertain the predominant GRD symptom types, GRD incidences and severity, farmers&rsquo; knowledge and their GRD coping mechanisms, the current groundnut seed system and farming practices. Data were analysed using SPSS and Chi-square tests of association. Mean GRD severity scores were geo-referenced and plotted on the Uganda map. Most respondent (52%) were females. Other than Northern Uganda, most regions grow groundnut landraces. Major seed sources were home saved and marketed. Thirty six percent of farmers grew groundnuts after cereals as recommended. All the farmers sampled knew about and had seen both rosette symptoms types, which were more visible during the second rains. A whole 42% of the farmers have no coping mechanism against GRD. The current knowledge of GRD did not have a significant effect on its management, seed source, varieties grown or gender of the farmers. The green rosette type predominates, making Uganda a green rosette belt. &nbsp; Key words: Arachis hypogaea L., groundnut rosette virus, green rosette, yellow rosette.

Groundnut rosette disease virus complex: Biology and molecular biology

Publisher Summary This chapter focuses on the cause of rosette disease of groundnut in which the umbravirus groundnut rosette virus (GRV) depends on groundnut rosette assistor virus (GRAV) for transmission by Aphis craccivora . The GRV/GRAV complex is, especially interesting, because the main cause of rosette disease symptoms in groundnut is a third component, a satellite RNA, which depends on GRV for its replication but on GRAV for its encapsidation and transmission by aphids. There are two major symptom variants, chlorotic rosette and green rosette with considerable variation within each type. Both the chlorotic and green forms of the disease make plants severely stunted, with shortened internodes and reduced leaf size. In chlorotic rosette, the leaves are bright yellow, usually with a few green islands. The biological properties of GRAV, which replicates autonomously in plants, are typical of a luteovirus and GRAV is therefore classified within the family Luteoviridae .

First Report of the Green Rosette Variant of Groundnut Rosette Disease in Kenya.

Groundnut (Arachis hypogaea L.) is an important food crop in sub-Saharan Africa. One of the major production constraints is groundnut rosette disease, which is caused by a complex of two viruses, groundnut rosette assistor luteovirus (GRAV) and groundnut rosette umbravirus (GRV) together with the associated satellite RNA (satRNA) (1). Two main forms of the disease have been described: chlorotic and the green rosette. Variants of the satRNA have been shown to be largely responsible for the different forms of the disease (1). Chlorotic rosette has been the predominant form in all of sub-Saharan Africa while green rosette has been reported in the western and southern regions of Africa (2). During the 1997-1998 crop season, disease surveys conducted in Kenya showed the incidence of the rosette disease in farmers' fields to be 24 to 40% in a total of 23 fields surveyed in the western regions of the country (Homabay, Kendubay, Kisumu) and 30% in 8 fields sampled in the Rift Valley (Cheplamus, Marigat) regions. Representative peanut plants showing rosette symptoms were analyzed for the presence of GRV by reverse transcription polymerase chain reaction (RT-PCR). With primers specific to a portion of ORF4 of GRV RNA (3), RT-PCR gave a product of expected size (approximately 300 bp). The PCR product was cloned in pGEM-T vector and sequenced. The sequenced region showed 89% nucleotide sequence identity with published GRV sequences. Green rosette was observed on groundnut cultivars Nyaela Red and Homabay Local in the Kendu Bay region. The incidence of the green rosette was 5.3% of the plants with rosette symptoms.

Different variants of the satellite RNA of groundnut rosette virus are responsible for the chlorotic and green forms of groundnut rosette disease*

SUMMARYGroundnut plants with symptoms of rosette disease contain groundnut rosette virus (GRV), but GRV is transmitted by Aphis craccivora only from plants that also contain groundnut rosette assistor virus (GRAV). Two main forms of rosette disease are recognised, ‘chlorotic rosette’ and ‘green rosette’. GRV cultures invariably possess a satellite RNA and this is the major cause of rosette symptoms: satellite‐free isolates derived from GRV cultures from Nigerian plants with chlorotic or green rosette, or from Malawian plants with chlorotic rosette, induced no symptoms, or only transient mild mottle or interveinal yellowing, in groundnut. When the satellite RNA species from GRV cultures from Nigerian green or Malawian chlorotic rosette were reintroduced into the three satellite‐free isolates in homologous and heterologous combinations, the ability to induce rosette symptoms was restored and the type of rosette induced was that of the cultures from which the satellite RNA was derived. Thus different forms of the satellite are responsible for the different forms of rosette disease. Other forms of the satellite induce only mild chlorosis or mottle symptoms in groundnut. Individual plants may contain more than one form of the satellite, and variations in their relative predominance are suggested to account for the variable symptoms (ranging from overall yellowing to mosaic) seen in some plants graft‐inoculated with chlorotic rosette.

A Simple Procedure to Detect a dsRNA Associated with Groundnut Rosette

AbstractA low‐molecular weight double‐stranded (ds) RNA [900 base pairs (bp)] associated with groundnut rosette disease can be used as a diagnostic tool. A simple procedure has been developed that is rapid, reliable, and requires only standard electrophoresis equipment and ultraviolet light for detection of nucleic acid bands. Using this procedure, the dsRNA was detected only in groundnut plants with green rosette of chlorotic rosette symptoms. It was not found in uninoculated groundnut plants, in symptomless groundnut plants with groundnut rosette assistor virus, or in groundnut plants infected with several other known groundnut viruses. In studies with northern blots of extracts from rosette‐diseased and healthy plants, 5′‐endlabeled dsRNA only hybridized to a 900 bp dsRNA from diseased plants. The 900 bp dsRNA was not infectious and its origin remains obscure.

Aphid Transmission of the Viruses Causing Chlorotic Rosette and Green Rosette Diseases of Peanut in Nigeria

Aphid Transmission of the Viruses Causing Chlorotic Rosette and Green Rosette Diseases of Peanut in Nigeria

Viruses associated with chlorotic rosette and green rosette diseases of groundnut in Nigeria*

SUMMARYGroundnut (Arachis hypogaea) plants from Nigeria with chlorotic rosette disease contained a manually transmissible virus, considered to be a strain of groundnut rosette virus (GRV(C)). GRV(C) infected nine out of 32 species in three out of nine families. It caused local lesions without systemic infection in Chenopodium amaranticolor, C. murale and C. quinoa, and systemic symptoms in Glycine max, Nicotiana benthamiana, N. clevelandii and Phaseolus vulgaris as well as in groundnut. Some ‘rosette‐resistant’ groundnut lines were also infected. GRV(C) was transmitted by Aphis craccivora, but only from groundnut plants that were also infected with an aphid‐transmissible second virus, which was not manually transmissible and was considered to be groundnut rosette assistor virus (GRAV). Plants infected with GRAV contained isometric particles c. 25 nm in diameter which were detectable by immunosorbent electron microscopy on grids coated with antisera to several luteoviruses, especially with antisera to bean leaf roll, potato leafroll and beet western yellows viruses. No virus‐like particles were observed in extracts from plants infected with GRV(C) alone.A single groundnut plant obtained from Nigeria with symptoms of green rosette contained luteovirus particles, presumed to be of GRAV, and yielded a manually transmissible virus that induced symptoms similar to those of GRV(C) in C. amaranticolor but gave only mild or symptomless infection of N. benthamiana and N. clevelandii. It was considered to be a strain of GRV and designated GRV(G).

Properties and partial purification of infective material from plants containing groundnut rosette virus*

SUMMARYGroundnut plants with chlorotic rosette disease contain a manually transmissible virus, groundnut rosette (GRV), which is also transmitted in the persistent (circulative) manner by aphids (Aphis craccivora), but only from plants that are co‐infected with a manually non‐transmissible luteovirus, groundnut rosette assistor virus (GRAV). Strains of GRV from plants with chlorotic or green forms of rosette are called GRV(C) and GRV(G) respectively. An isolate of GRV(C) from Nigeria remained infective in Nicotiana clevelandii leaf extracts for 1 day at room temperature and for 15 days at 4d̀C, but lost infectivity after 1 day at ‐20d̀C or after dilution to 10‐‐4. Its infectivity and longevity in vitro were not altered by addition of 1 mg/litre bentonite to the extraction buffer. Infectivity in leaf extracts was abolished by treatment with 50% (v/v) ether, 10% (v/v) chloroform or 8% (v/v) n‐butanol, but not by treatment for 30 min with RNase A at up to 100 ng/ml. In attempts to purify GRV(C), nearly all the infectivity from N. clevelandii extracts was found in the pellets from centrifugation at 65 000 g for 1. 5 h; infectivity also occurred in a cell membrane fraction that collected at the top of a 30% sucrose ‘cushion’ containing 4% polyethylene glycol and 0.2 M NaCI. However, no virus‐like particles were found in either type of preparation by electron microscopy.Nucleic acid preparations made directly from GRV(C)‐infected N. clevelandii leaves were very infective; this infectivity was totally inactivated by treatment for 30 min with RNase A at 10 ng/ml in buffers of both low and high ionic strength and was therefore attributed to ssRNA. When nucleic acid preparations were electrophoresed in gels no virus‐specific bands were visible but the position of the infectivity indicated that the infective ssRNA has an apparent mol. wt of c. 1.55 × 106. A similar mol. wt was indicated by the rate of sedimentation of the infective ssRNA in sucrose gradients. Preparations of dsRNA made from GRV(C)‐infected N. clevelandii leaves contained a species of mol. wt c. 3.0 × 106; in addition some dsRNA preparations contained an abundant component of mol. wt c. 0.6 × 106 together with several other components of intermediate mol. wt. Similar patterns of bands were observed in dsRNA preparations made from Nigerian‐grown groundnut material infected with GRV(C) alone, or with GRV(C) + GRAV, or with GRV(G) + GRAV.The properties of GRV closely resemble those of two other viruses that depend on luteoviruses for transmission by aphids, carrot mottle virus and lettuce speckles mottle virus.

Groundnut rosette and its assistor virus

SUMMARYChlorotic rosette from Malawi (isolate CR1), passed through Stylosanthes gracilis and S. juncea, was not subsequently transmissible from groundnuts (Arachis hypogaea) by Aphis craccivora or A. gossypii, but with S. mucronata transmissibility was occasionally regained after a period of time. Aphid transmissibility was similarly lost after passage of two isolates (a chlorotic rosette from Rhodesia, CR2, and a green rosette from Nigeria, GR) through soybean (Soja max) and after manual inoculation to groundnuts. Groundnut plants that remained symptomless after exposure to rosette infection by aphids often contained a virus that restored aphid transmissibility when introduced into groundnuts containing the vectorless virus from that isolate. Groundnut rosette disease therefore consists of a symptom‐inducing virus that we call groundnut rosette virus (GRV) and a symptomless assistor virus (GRAV) that must be present for aphid transmission. The interactions between the GRV and GRAV of chlorotic and green rosette, and their transmission by different vector races, are described.