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).     

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.     

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.     

Studies on the transmission of groundnut rosette virus by Aphis craccivora Koch

Four strains of groundnut rosette virus were transmitted by a race of Aphis craccivora (Koch) from groundnut in Nigeria. Two of these strains, both from East Africa, were transmitted only by A. craccivora from Kenya. A fifth isolate, from Nigeria, was not transmissible by either race. The two races of aphids have been shown elsewhere to be distinct biotypes. Most A. craccivora needed longer than 24 h feeding on infected groundnuts to acquire virus, and many needed 2–3 days of feeding on healthy plants to cause infection, even after several days on infected plants. The delays partly reflect the slow uptake of virus and possibly a period needed for virus multiplication in aphid tissue but some is lost through resistance of the test plants to infection. In consecutive feeding experiments Natal Common variety could be infected soon after aphids had left the source of virus, but a more resistant Nigerian variety sometimes needed several more days. The frequency of inoculation by aphids, or the concentration of virus in the inocula or both, increased with time, but the times at which aphids were able to infect plants was also dependent on variety.     

A Simple Procedure to Detect a dsRNA Associated with Groundnut Rosette

A 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.     

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.     

A variant of the satellite RNA of groundnut rosette virus that induces brilliant yellow blotch mosaic symptoms in Nicotiana benthamiana

Some Malawian cultures of groundnut rosette virus (GRV) give rise to variants that, although still causing symptoms of the chlorotic type of rosette in groundnut, induce brilliant yellow blotch mosaic symptoms, instead of the usual veinal chlorosis and mild mottle, in Nicotiana benthamiana. One such isolate (YB) induced the formation in infected plants of a 0.9 kbp dsRNA having extensive sequence homology with molecules of similar size in other naturally occurring isolates of GRV. These dsRNA molecules were shown to be double-stranded forms of single-stranded satellite RNA molecules. Experiments in which the satellite was removed from and restored to isolate YB, or exchanged with those from other GRV isolates, showed that it carries the determinant for yellow blotch mosaic symptoms. Plants inoculated with the 0.9 kbp dsRNA (denatured or undenatured) developed yellow blotch mosaic even when the satellite-free GRV helper was not inoculated until 11 days later. The satellite RNA is therefore a very stable molecule. Prior infection of N. benthamiana with a GRV isolate containing a normal form of the satellite protected against expression of yellow blotch mosaic symptoms when the plants were later inoculated with isolate YB, whereas prior infection with satellite-free isolates did not. This provides a simple method of determining whether a GRV isolate has an associated satellite RNA. The YB satellite seems to be a newly recognised variant additional to those known to cause the chlorotic, green and other forms of groundnut rosette disease.     

Host range and some properties of groundnut rosette virus

Two isolates of groundnut rosette virus from East Africa (GRVE₁ and GRVE₂) and from West Africa (GRVW₁ and GRVW₂) were transmitted by Aphis craccivora obtained from West Africa. A third isolate from West Africa (GRVW₃) was not transmitted by A. craccivora from three widely separated sources. GRVW₁, GRVW₂ and GRVW₃ caused leaf-symptoms in groundnut of a mosaic pattern in light and dark green. GRVE₁ and GRVE₂ caused chlorosis or chlorosis and leaf distortion as well as mosaic symptoms. Groundnut plants with GRVW₁ could not be infected by means of aphids with GRVE₁, and GRVE₁ gave similar protection against GRVW₁, which suggests that they are strains of the same virus. All isolates were transmissible manually from groundnut to groundnut (Arachis hypogea), Trifolium incarnatum and T. repens, and caused systemic infection. Inoculated Nicotiana clevelandii and N. rustica developed symptoms but virus could not be recovered from them. Chenopodium amaranticolor, C. hybridum and C. quinoa showed local lesions on inoculated leaves. Virus could be acquired by aphids from groundnut or Trifolium repens infected by means of aphids, but not from those infected by manual inoculation. Virus could not be recovered from T. incarnatum manually or by aphids, but was transmitted by cleft-grafting from clover to groundnut. Saps extracted in borax buffer plus zinc sulphate at pH 9 from plants infected with GRVW₁ and GRVE₁ remained infective at 18° C. for 1 week, and at — 20° C. for up to 4 weeks. Virus could be recovered from frozen leaves. Buffered saps lost infectivity when heated above 50° C. for 10 min.; most were still infective when diluted 1/10 and some at 1/100. Electron micrographs of partially purified preparations contained spherical particles 25–28 mμ in diameter. There were usually only about five per microscope field and they resembled those of some other viruses.     

Detection of a Luteovirus in Groundnut Rosette Diseased Groundnuts (Arachis hypogaea) by Enzyme-linked Immunosorbent Assay and Immunoelectron Microscopy

In groundnut rosette diseased groundnut plants collected near Zaria, Nigeria, a luteovirus was detected by ELISA and ISEM. In ELISA only beet western yellows virus antiserum reacted, while in ISEM luteovirus particles were trapped by antisera beet western yellows virus, potato leafroll virus, pea leafroll virus and barley yellow dwarf virus. The data are in agreement with the interpretation that the assistor of groundnut rosette virus is possibly a member of the luteovirus group.     

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.     

Transmission and differentiation of six viruses infecting hogweed (Heracleum sphondylium) in Scotland

Six viruses, code-named HV1-HV6, were transmitted manually and/or by aphids (Cavariella spp. from symptomless wild plants of hogweed (Heracleum sphondylium) in Scotland. HV1 was identified as parsnip yellow fleck virus (PYFV); anthriscus yellows virus, on which it depends for transmission by aphids, was presumably also present in the hogweed plants. HV2 was transmitted manually and by aphids and had very flexuous filamentous particles c. 700–750 nm long; it has affinities with the closteroviruses, and the name heracleum latent virus is proposed. HV3, HV4 and HV5 were transmitted manually, HV3 and HV5 also by aphids, but their particle morphology is unknown. HV6 was transmitted only by aphids and has very flexuous particles up to 1400 nm long; it is presumably a closterovirus distinct from HV2. All the viruses infected cultivated umbelliferous species experimentally but only PYFV is known to infect umbelliferous crops.     

Epidemiology of groundnut rosette virus disease: current status and future research needs@

Rosette is the most destructive virus disease of groundnut in sub-Saharan Africa. It is caused by a complex of three agents, namely groundnut rosette assistor virus, groundnut rosette virus and its satellite RNA. The disease appears to be indigenous to Africa as it has not been recorded elsewhere. Thus rosette represents a new-encounter situation as the disease is thought to have spread to the introduced groundnut from indigenous host plants. Rosette has been known since 1907 and much information has been obtained on the main features of the disease, viz. its biology, transmission, viral aetiology and diagnosis, and the impact of chemical control of the aphid vector, cultural practices and virus-resistant varieties on disease management. However, there are still many gaps in the available knowledge, especially the reasons for the large and unpredictable fluctuations in the incidence and severity of rosette disease throughout sub-Saharan Africa. Three unresolved issues of particular importance concern the nature of the primary source(s) of inoculum, the means of survival of virus and vector during unfavourable periods, and the distances over which the aphid vector can disperse and disseminate virus. Now that the aetiology of the disease is understood and diagnostic tools have been developed, the time is opportune for new initiatives in understanding the ecology and epidemiology of rosette. Substantial progress can be made by developing a co-ordinated multi-disciplinary research programme and making full use of the latest techniques, approaches and experience gained elsewhere with other insect-borne viruses. This information would help to explain the sporadic disease epidemics that cause serious crop losses and sometimes total crop failure, and would also facilitate the development of disease forecasting methods and sustainable integrated disease management strategies.     

Further evidence on the nature of the dependence of carrot mottle virus on carrot red leaf virus for transmission by aphids

Preparations were made from chervil plants doubly infected with carrot mottle virus (CMotV) and its helper virus, carrot red leaf (CRLV), on which it depends for transmission by the aphid Cavariella aegopodii, by the procedure developed previously for CRLV. The preparations contained 25 nm isometric particles which were indistinguishable from those of CRLV but possessed aphid-transmissible infectivity of both viruses and manually transmissible infectivity of CMotV. Only one sedimenting and buoyant density component was detected. The manually transmissible CMotV infectivity was resistant to freezing and to organic solvents, treatments that destroyed the CMotV infectivity in extracts from singly infected plants. The aphid-transmissible CMotV infectivity in preparations from CRLV/ CMotV-infected plants, and that in extracts from CRLV/CMotV-carrying C. aegopodii, was abolished by treatment with CRLV antiserum but not with normal serum. These results show that transmission of CMotV by C. aegopodii is dependent on the packaging of its RNA in coats composed partially or entirely of CRLV particle protein. The aphid Myzus persicae does not transmit CRLV or CMotV from plants mixedly or singly infected with these viruses but it is a vector of beet western yellows virus (BWYV) and potato leafroll virus (PLRV) and it transmitted CMotV from plants that also contained either of these viruses. This suggests that the coat proteins of BWYV and PLRV can substitute for that of CRLV in packaging CMotV nucleic acid and thereby confer on it their own vector specificities.     

TRANSMISSION OF GROUNDNUT ROSETTE VIRUS

The rosette virus was transmitted to groundnut plants, if previously etiolated, bymechanical inoculation of juice with 'Celite' addition; but only a small proportion of the inoculations succeeded.
Aphis craccivora (Koch), the known vector, transmitted the virus by feeding on germinating groundnut seeds; and from this fact we developed an experimental technique that is convenient and flexible.
Different races of this species appeared to vary in inherent efficiency in transmission, and one failed ever to transmit. Within races that could transmit, all larval forms and alate and apterous adults might transmit; but alatae were sometimes significantly more efficient than apterae, and at other times the converse held. A field experiment showed that wingless forms, moving over the soil surface, might play a predominant part in secondary spread around a rosetted plant.
Comparative tests with groups of I, 2, 3 and 4 infective aphids supported the hypothesis that infections by this vector are individual and independent.
Single aphids, tested for 24 hr. on 10 successive days without access to an outside source of virus, might infect on any day up to the tenth. Similar results were obtained in a succession of I hr. tests on a single day. In the infected seed the virus rapidly became available to feeding aphids; previously non-infective aphids acquired the virus by feeding on a seed during the third day from the first exposure of this seed to infective aphids.     

幽影病毒引起的几种主要植物病害

幽影病毒的基因组不编码外壳蛋白,不形成通常的病毒粒体结构。这类病毒往往和黄症病毒复合侵染引起植物病害,蚜虫传播是病害在田间传播流行的主要方式。对幽影病毒引起的胡萝卜杂色矮缩病、花生丛簇病以及烟草丛顶病等几种主要病害的症状、发生与危害、病原物特性以及病害的控制等进行了综述。     

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.     

VIRUSES CAUSING ROSETTE AND OTHER DISEASES IN GROUNDNUTS

From plants with a form of groundnut rosette disease, characterized by discrete areas of green and chlorotic tissue on the leaflets and here designated 'mosaic rosette', a virus was separated that produced only a mild mottle or sometimes a mottle with rare chlorotic flecks. It was separated by leaf grafts, by mechanical inoculation and by Aphis craccivora .
Plants inoculated simultaneously with the mottle virus and normal rosette virus usually developed the mosaic-rosette symptoms. When the mottle virus was introduced 14–35 days before the rosette virus, the plants failed to develop the severe chlorosis of rosette; the mottle virus thus protected the plant from rosette, and this was true whether the rosette virus was inoculated by aphids or by grafting.
Plants showing two other forms of mild mottle were collected in the field; viruses from them were readily transmitted by grafting or by mechanical inoculation, but not by A. craccivara . In plant-protection tests with one of these, it failed to protect plants from developing chlorotic symptoms when later inoculated with the rosette virus, although a form of interaction was evident and the doubly-infected plant was less severely chlorotic and less stunted than one infected with the rosette virus alone.     

THE EFFECT OF DIFFERENT HOST PLANTS OF POTATO VIRUS C IN DETERMINING ITS TRANSMISSION BY APHIDS

A stock of potato virus C derived from Edgecote Purple potatoes in 1945 was not then transmitted by aphids, although more than 2000 aphids were used in conditions optimal for transmitting the serologically related potato virus Y. This stock of virus C has been propagated continuously since, by manual inoculation in a series of Nicotiana glutinosa and N. tabacum , and in 1955 it was transmitted by the aphid Myzus persicae (Sulz.): about one in twenty of the aphids transmitted it compared with one in two for potato virus Y.
Virus C derived from the Edgecote Purple potatoes in 1955 was not transmitted by aphids; both stocks of virus C produced only local lesions in Majestic potato leaves, and gave similar symptoms in tobacco.
When inoculated to Majestic potatoes and then returned to tobacco plants, potato virus C usually ceased to be aphid transmitted and did not recover this property in any of the subsequent subcultures.
Transmission from stock by aphids did not isolate a strain of virus C which was any more readily transmitted by aphids, indeed, for the first two or three subcultures, aphids usually transmitted more readily from plants inoculated manually. But the few isolates which remained aphid transmissible, after a second passage through potato, were rather readily transmitted.
These results suggest that the ability of a virus to be aphid transmitted is, at least in part, determined by the host plant in which it is multiplying, but the nature of the changes which determine this ability are unknown.     

Purification, properties and transmission of parsnip yellow fleck, a semi-persistent, aphid-borne virus

Parsnip yellow fleck virus (PYFV) is the commonest cause of virus-like symptoms in parsnip plants in Britain: it is sap-transmissible but systemically infects few species outside the Umbelliferae. It has isometric particles 29–31 mμ in diameter, a sedimentation coefficient of 167s, and loses infectivity in sap after dilution to 10^-3-10^-4, heating for 10 min at 57·5–65°C, or storage at room temperature for 4–7 days. Two isolates, from parsnip and Anthriscus sylvestris respectively, are only distantly serologically related. The aphid Cavariella aegopodii transmits PYFV in a semi-persistent manner from A. sylvestris but not from parsnip. Transmission by aphids apparently depends on the presence in A. sylvestris or other source plants of a second virus, anthriscus yellows (AYV), which is persistent in the vector and not manually transmissible. PYFV was therefore not transmitted by aphids from manually inoculated plants or from parsnip or other plants immune to AYV. In controlled experiments, C. aegopodii transmitted PYFV (both A. sylvestris and parsnip isolates) from chervil plants inoculated separately with PYFV and AYV, but not from plants inoculated only with PYFV.