Groundnut rosette and its assistor virus

Chlorotic 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.     

Additional sources of resistance to groundnut rosette disease in groundnut germplasm and breeding lines

Groundnut rosette, a virus disease of groundnut (Arachis hypogaea) transmitted by the aphid, Aphis craccivora Koch, reduces yield in susceptible cultivars by 30–100%. Additional sources were sought in germplasm accessions involving 2301 lines from different sources and from 252 advanced breeding lines derived from crosses involving earlier identified sources of resistance to rosette. The lines were evaluated in field screening trials using an infector row technique during 1996 and 1997 growing seasons. Among the germplasm lines, 65 accessions showed high levels of resistance while 134 breeding lines were resistant. All rosette disease resistant lines were susceptible to groundnut rosette assistor virus. This work identified germplasm and breeding lines that will contribute to an integrated management of groundnut rosette disease. These new sources also provide an opportunity to eliminate yield losses due to the rosette disease.     

The nature of the resistance in groundnut to rosette disease

Groundnut rosette disease is caused by a complex of three agents, groundnut rosette virus (GRV) and its satellite RNA, and groundnut rosette assistor virus (GRAV); the satellite RNA is mainly responsible for the disease symptoms. Groundnut genotypes possessing resistance to rosette disease were shown to be highly resistant (though not immune) to GRV and therefore to its satellite RNA, but were fully susceptible to GRAV.     

The Effect of Groundnut rosette assistor virus on the Agronomic Performance of Four Groundnut (Arachis hypogaea L.) Genotypes

The effect of Groundnut rosette assistor virus (GRAV), in the absence of the other two agents (Groundnut rosette virus and its satellite RNA) of the groundnut rosette disease virus complex, was evaluated on the agronomic performance of four groundnut (=peanut) genotypes (CG‐7, ICGV‐SM‐90704, JL‐24 and ICG‐12991) with different botanical characteristics. All genotypes infected with GRAV showed mild yellowing/chlorosis of leaves and the symptoms persisted throughout their growth period. ELISA absorbance values indicated lower amounts of GRAV antigen in ICGV‐SM‐90704 than in the other genotypes. The reduction in leaf area due to GRAV infection varied between 15.5% and 21.7%, whereas the plant height was decreased between 11.3% and 13.4% among the four genotypes. GRAV infection caused 28.4%, 16.9%, 21.7% and 25.5% reduction in the dry weight of haulms in CG‐7, ICGV‐SM‐90704, JL‐24 and ICG‐12991 respectively. Plants infected with GRAV showed greater reduction in seed weight in CG‐7 (52.2%), followed by JL‐24 (46.1%), ICG‐12991 (40.7%) and ICGV‐SM‐90704 (25.7%). These results provide evidence for the first time that GRAV infection, without GRV and sat RNA, affect plant growth and contribute to yield losses in groundnut.     

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

Groundnut (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).     

Mechanisms and diversity of resistance to insect pests in wild relatives of groundnut

The levels of resistance to insect pests in cultivated groundnut (Arachis hypogaea) germplasm are quite low, and therefore, we screened 30 accessions of Arachis spp. and 12 derived lines for resistance to insect pests under field and greenhouse conditions. Accessions belonging to Arachis cardenasii, Arachis duranensis, Arachis kempff-mercadoi, Arachis monticola, Arachis stenosperma, Arachis paraguariensis, Arachis pusilla, and Arachis triseminata showed multiple resistance to the leaf miner Aproaerema modicella, Helicoverpa armigera, Empoasca kerri, and to rust, Puccnia arachidis Speg., and late leaf spot, Cercosporidium personatum (Berk. et Curt.). Arachis cardenasii (ICG 8216), Arachis ipaensis (ICG 8206), A. paraguariensis (ICG 8130), and Arachis appressipila (ICG 8946) showed resistance to leaf feeding and antibiosis to Spodoptera litura under no-choice conditions. Six lines, derived from wild relatives, showed resistance to H. armigera and S. litura, and/or leaf miner. Plant morphological characteristics such as main stem thickness, hypanthium length, leaflet shape and length, leaf hairiness, standard petal length and petal markings, basal leaflet width, main stem thickness and hairiness, stipule adnation length and width, and peg length showed significant correlation and/or regression coefficients with damage by H. armigera, S. litura, and leafhoppers, and these traits can possibly be used as markers to select for resistance to these insect pests. Principal component analysis placed the Arachis spp. accessions into five groups, and these differences can be exploited to diversify resistance to the target insect pests in groundnut.     

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

Groundnut 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 large scale analysis of resistance gene homologues in<Emphasis Type="Italic"> Arachis</Emphasis>

Arachis hypogaea L., commonly known as the peanut or groundnut, is an important and widespread food legume. Because the crop has a narrow genetic base, genetic diversity in A. hypogaea is low and it lacks sources of resistance to many pests and diseases. In contrast, wild diploid Arachis species are genetically diverse and are rich sources of disease resistance genes. The majority of known plant disease resistance genes encode proteins with a nucleotide binding site domain (NBS). In this study, degenerate PCR primers designed to bind to DNA regions encoding conserved motifs within this domain were used to amplify NBS-encoding regions from Arachis spp. The Arachis spp. used were A. hypogaea var. Tatu and wild species that are known to be sources of disease resistance: A. cardenasii, A. duranensis , A. stenosperma and A. simpsonii. A total of 78 complete NBS-encoding regions were isolated, of which 63 had uninterrupted ORFs. Phylogenetic analysis of the Arachis NBS sequences derived in this study and other NBS sequences from Arabidopsis thaliana, Medicago trunculata , Glycine max , Lotus japonicus and Phaseolus vulgaris that are available in public databases This analysis indicates that most Arachis NBS sequences fall within legume-specific clades, some of which appear to have undergone extensive copy number expansions in the legumes. In addition, NBS motifs from A. thaliana and legumes were characterized. Differences in the TIR and non-TIR motifs were identified. The likely effect of these differences on the amplification of NBS-encoding sequences by PCR is discussed.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Communicated by M.-A. Grandbastien     

Identification of resistance to Peanut bud necrosis virus (PBNV) in wild Arachis germplasm

Eighty three wild Arachis germplasm accessions, belonging to 24 species of five sections and one natural hybrid derivative of a cross between the cultivated and a wild Arachis species, were evaluated along with a susceptible groundnut cultivar for resistance to Peanut bud necrosis virus (PBNV) in a replicated field trial at ICRISAT, Patancheru, India. Thirty days after sowing, the percentage of infected plants were recorded for all the accessions and subsequently young leaflets from all these accessions were tested for the presence of the virus by enzyme linked immunosorbent assay (ELISA). One accession each of A. benensis and A. cardenasii, and two accessions of A. villosa, in the section Arachis, two accessions of A. appressipila in the section Procumbentes, and one accession of A. triseminata under section Triseminatae were not infected by PBNV. These seven field‐resistant accessions were tested under glasshouse conditions for virus resistance by mechanical sap inoculations. One accession of A. cardenasii and two accessions of A. villosa did not show systemic infection. Similarly, in another glasshouse test, where 13 A. cardenasii accessions of section Arachis were evaluated, two accessions did not show systemic infection. In all these resistant accessions, the inoculated leaves showed infection, but the systemic leaves did not show the presence of virus in spite of repeated mechanical sap inoculations. So, the resistance in these accessions appears to be due to a block in systemic movement of the virus. To our knowledge this is the first report on the identification of resistance to PBNV in wild Arachis species. Since both A. cardenasii and A. villosa are the progenitors of cultivated groundnut and can be hybridised with the latter, the resistant accessions are being utilised in conventional breeding programmes to transfer PBNV resistance to widely adapted groundnut cultivars.     

Use of monoclonal antibody to potato leafroll virus for detecting groundnut rosette assistor virus by ELISA*

Three of 10 monoclonal antibodies (MAbs) produced to potato leafroll luteovirus (PLRV) were found to react in triple antibody sandwich ELISA (TAS-ELISA) with groundnut rosette assistor luteovirus (GRAV), though none reacted with four other luteoviruses (barley yellow dwarf, bean leaf roll, beet western yellows or carrot red leaf)- The most effective PLRV MAb, SCR 6, was used in TAS-ELISA to detect isolates of GRAV from groundnut plants with chlorotic, green and mosaic forms of rosette from Nigeria and Malawi. The test also detected GRAV in extracts of single Aphis craccivora.     

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

Groundnut 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 4d?C, but lost infectivity after 1 day at -20d?C 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.     

Identification of Fungus Resistant Wild Accessions and Interspecific Hybrids of the Genus Arachis

Peanut, Arachis hypogaea L., is a protein-rich species consumed worldwide. A key improvement to peanut culture involves the development of cultivars that resist fungal diseases such as rust, leaf spot and scab. Over three years, we evaluated fungal resistance under field conditions of 43 wild accessions and three interspecific hybrids of the genus Arachis, as well as six A. hypogaea genotypes. In the first year, we evaluated resistance to early and late leaf spot, rust and scab. In the second and third years, we evaluated the 18 wild species with the best resistance scores and control cultivar IAC Caiapó for resistance to leaf spot and rust. All wild accessions displayed greater resistance than A. hypogaea but differed in their degree of resistance, even within the same species. We found accessions with as good as or better resistance than A. cardenasii, including: A. stenosperma (V15076 and Sv 3712), A. kuhlmannii (V 6413), A. kempff-mercadoi (V 13250), A. hoehnei (KG 30006), and A. helodes (V 6325). Amphidiploids and hybrids of A. hypogaea behaved similarly to wild species. An additional four accessions deserve further evaluation: A. magna (V 13751 and KG 30097) and A. gregoryi (V 14767 and V 14957). Although they did not display as strong resistance as the accessions cited above, they belong to the B genome type that is crucial to resistance gene introgression and pyramidization in A. hypogaea.     

Sources of resistance to groundnut rosette disease in global groundnut germplasm*

About 6800 groundnut germplasm accessions originating from South America, Africa, and Asia were evaluated for resistance to rosette disease using an infector row technique between the 1990/91 and 1996/97 growing seasons. Of these, 116 germplasm accessions, including 15 short-duration Spanish types, have shown high levels of resistance to rosette disease. A high percentage of these resistant accessions were from West Africa and a few were from Asia and southern Africa. Only one out of 1400 accessions from South America showed resistance to rosette disease. All disease-resistant accessions were susceptible to groundnut rosette assistor virus. This is the first report to identify sources of resistance to rosette disease in groundnut germplasm from Asia and South America. These additional sources of resistance provide an opportunity to broaden the genetic base of resistance to rosette disease. The origins of rosette resistance in groundnut are discussed.     

A Study of Gene Expression in the Nematode Resistant Wild Peanut Relative, Arachis stenosperma, in Response to Challenge with Meloidogyne arenaria

Peanut (Arachis hypogaea) is amongst the most important legume crops in the world. One of its main yield constraints is the root-knot nematode Meloidogyne arenaria. A number of wild Arachis species, including A. stenosperma, are resistant to nematodes, and are a potential source of new resistance alleles for cultivated peanut. Using in silico subtraction of ESTs and macroarray analysis, we identified genes differentially expressed in A. stenosperma roots during its resistance response to M. arenaria. The three most differentially expressed genes [Auxin Repressed Protein (AsARP), Cytokinin Oxidase (AsCKX) and Metallothionein Type 2 (AsMET2)] were further analyzed using northern-blot and showed distinct expression profiles in the resistant A. stenosperma and susceptible A. hypogaea, both after, and sometimes even before, challenge with nematodes. Of the three most differentially expressed genes, AsARP and AsCKX are potentially involved in plant hormonal balance, and AsMET2 may be related to the reactive oxygen reaction triggered by the hypersensitive response (HR).     

Isolation and characterization of microsatellite loci from the forage species Arachis pintoi (Genus Arachis)

Arachis pintoi is an alternative to forage production in the tropics. Its germplasm comprises more than 150 accessions, that could be used to improve it. Our objective was the isolation and characterization of microsatellite loci in A. pintoi to be used to molecular evaluation of this germplasm and of A. repens (section Caulorrhizae ). Seven loci were analyzed using five accessions of A. repens and 20 accessions of A. pintoi . The high variation found makes clear the high potential of this marker in genetic studies in these species. The developed markers showed total transferability to A. repens .     

Diversity of seed storage protein patterns in wild peanut (Arachis,Fabaceae) species

55 accessions of wild peanuts (Arachis spp.) introduced from South America were analyzed for seed storage protein composition using SDS-PAGE electrophoresis. The objectives of the study were to evaluate variability within sect.Arachis and to classify taxa based on protein composition. 25 different band positions were resolved. Individual accessions had 11 to 18 bands which included the conarachin region (MW > 50 kD), two to five bands in the acidic arachin region (MW 38–49.9 kD), three to seven in the intermediate MW region (23 to 37.9 kD), two to five bands in the basic arachin region (18–22.9 kD), and one to three bands in the low MW protein region (14–17.9 kD). These data were utilized in a principal coordinate analysis based on the matrix of genetic distances between all pairs of the 55 accessions. Several groups of accessions conformed to expected species classification includingA. batizocoi, A. stenosperma, andA. monticola; whileA. duranensis, A. cardenasii, A. helodes, andA. correntina did not form good groups. The study showed that great diversity exists for protein profiles and seed storage proteins have potential for aiding species classification and for serving as markers for interspecific hybridization studies.     

Host specificity among five species in the cowpea cross-inoculation group

Summary In a cross-inoculation experiment using crushed nodules from cowpea (Vigna unguiculata), groundnut (Arachis hypogea), bambara groundnut (Voandzeia subterranea), lima bean (Phaseolus lunatus) and soybean (Glycine max.), it was found that soybean did not nodulate with Rhizobia from any of the other species whilst its Rhizobia nodulated with all species. Cowpea and lima bean, on the other hand, nodulated with Rhizobia from all species, but their Rhizobia nodulated only with each other. Groundnut and bambara groundnut nodulated with Rhizobia from all species except cowpea and lima bean, and their Rhizobia also nodulated with all species except soybean.     

Abundant Microsatellite Diversity and Oil Content in Wild Arachis Species

The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil content is becoming increasingly important. Wild Arachis species have been reported to harbor genes for many valuable traits that may enable the improvement of cultivated Arachis hypogaea, such as resistance to pests and disease. However, only limited information is available on variation in oil content. In the present study, a collection of 72 wild Arachis accessions representing 19 species and 3 cultivated peanut accessions were genotyped using 136 genome-wide SSR markers and phenotyped for oil content over three growing seasons. The wild Arachis accessions showed abundant diversity across the 19 species. A. duranensis exhibited the highest diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique alleles were detected in the species studied. A. rigonii exhibited the largest number of unique alleles (75), indicating that this species is highly differentiated. AMOVA and genetic distance analyses confirmed the genetic differentiation between the wild Arachis species. The majority of SSR alleles were detected exclusively in the wild species and not in A. hypogaea, indicating that directional selection or the hitchhiking effect has played an important role in the domestication of the cultivated peanut. The 75 accessions were grouped into three clusters based on population structure and phylogenic analysis, consistent with their taxonomic sections, species and genome types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the close relationship between these two diploid wild species and the cultivated peanut. Considerable phenotypic variation in oil content was observed among different sections and species. Nine alleles were identified as associated with oil content based on association analysis, of these, three alleles were associated with higher oil content but were absent in the cultivated peanut. The results demonstrated that there is great potential to increase the oil content in A. hypogaea by using the wild Arachis germplasm.     

Purification and particle properties of groundnut rosette assistor virus and production of a specific antiserum*

Purified preparations of the luteovirus, groundnut rosette assistor virus (GRAV), were made by treatment of groundnut leaf extracts with cellulase, followed by sucrose density gradient centrifugation. Yields of virus particles were about 0·5-1·0 mg/kg leaf material. The preparations contained isometric particles c. 28 nm in diameter with a sedimentation coefficient (s_{20, w}) of 115 S, a buoyant density in Cs₂SO₄ of 1·34 g/cm³, and A₂₆₀/A₂₈₀ of 1·86. The particles contained a single species of nucleic acid (presumably RNA), of mol. wt 2·09 × 10⁶and with no detectable polyadenylate sequence, and a single protein species, of mol. wt 24 × 10³. An antiserum produced in a rabbit had a titre of 1/256 in gel diffusion tests and detected GRAV in leaf extracts by ELISA. GRAV particles reacted in F(ab')₂-ELISA and immunosorbent electron microscopy (ISEM) tests with antisera to bean leaf roll, potato leafroll and tobacco necrotic dwarf luteoviruses, but did not react with antisera to carrot red leaf luteovirus.