Ann. appl. Biol. (1973), 74, 171-179 With I plate Printed in Great Britain

Peanut mottle virus in East Africa

BY K. R. BOCK East African AgricuZture and Forestry Research Organization,

P. 0. Box 30 I 48, Nairobi, Kenya

(Accepted 3 January 1973)

SUMMARY

A very common and widespread virus pathogen of groundnut and soybean in East Africa was identified as peanut mottle virus (PnMV)" on the basis of particle morphology, serology, host range and reaction, transmission and physical properties.

Virus concentration adequate for serological tests was obtained from cow- pea (Vigna unguinclata) cultured at 27 "C but not at 23 "C. Purified prepara- tions from this source gave a single, specific light-scattering zone in sucrose density gradients.

PnMV was purified using 0.5 M sodium citrate buffer containing I % mercapto-ethanol ; an antiserum made against such preparations had a homologous titre of 1/8192.

Groundnut and soya isolates from N., N.E., N.W. and S. districts of Uganda, N.W. Tanzania, and W. and E. (coastal) districts of Kenya were serologically similar and varied, within narrow limits, in symptoms induced in certain groundnut and soya varieties. A serologically related but distinct virus was isolated from Voandxeia subterranea.

PnMV was not related serologically to any of ten viruses of the PVY group.

Glasshouse experiments simulating groundkeeper conditions in the field indicated 20% seed transmission in groundnut; PnMV was transmitted by Aphis craccivora in the non-persistent manner.

All twenty-one varieties and breeding lines of soybean tested were highly susceptible.

The prevalence of PnMV in East Africa and the reduction in yield caused in groundnut indicates the virus to be economically important, and groundnut and soybean improvement programmes should include routine PnMV sus- ceptibility tests.

INTRODUCTION

Although several virus diseases of groundnut (Arachis hypogaea L.) have been described from Africa (Brunt & Bonney, 1964; Hayes, 1932; Klesser, 1965; Storey & Ryland, 1957), none of the causal viruses has been characterized; their inter-relation- ships and affinities with other viruses are unknown. In East Africa, Storey & Ryland (1957), working with groundnut rosette disease, differentiated a rosette virus, a mottle virus which they concluded induced mosaic rosette symptoms in association with rosette virus, and two other 'mottle' viruses. Their basis of discrimination was con-

* Abbreviated to PnMV to avoid confusion with pea mosaic virus (normally abbreviated to PMV).

K. R. BOCK sistent symptom expression in the cultivar Natal Common, and none of the isolates was characterized on the basis of particle morphology, physical properties, host range and serology.

Detailed studies of viruses affecting groundnuts in East Africa were initiated during a virus disease survey of mainfood legumes. Although effort was directed towards groundnut rosette disease and Storey & Ryland's mottle viruses, another virus, which induced local necrotic lesions on Phaseolus vulgaris L., was very common and wide- spread in groundnut, and was easily isolated; 43 yo of field groundnut plants indexed were infected with this virus.

A similar virus was also isolated from soybean (Glycine max (L.) Merr.) in all areas surveyed. It was important to know the relationship of the many geographically distinct virus isolates to each other, and their relationship to viruses of groundnut and soya described from America and elsewhere. This paper reports the East African distribution, and the purification, properties and serological affinities of the virus, and its identification as peanut mottle virus (PnMV) (Kuhn, 1965), recorded for the first time in Africa.

MATERIALS AND METHODS

Collections were made in many areas of Uganda, Kenya and Tanzania of groundnut plants which showed rosette or mottle symptoms or were stunted. Test plants were dusted with carborundum prior to inoculation. Young leaves from field material were ground in 0,067 M-PO, buffer, pH 7.7, and the sap rubbed onto leaves of P. vulgaris. Single lesion isolations from subsequent necrotic local lesions were made to G. max cultivar HLS 541, a local selection. Similar isolations were made from virus-infected soya from field sources.

Virus isolates were maintained in G . max and Cassia occidentalis L. ; selected iso- lates were cultured at 27 "C in cowpea (Vigna ungukluta Walp.) for purification. Phaseolus vulgaris was used as a local lesion assay host. Test plants were grown in glasshouses at a mean temperature of 23 "C, and maintained insect free by the use of 20 % Vapona insecticide resin strips (2,~-dichlorovinyl dimethyl phosphate). Plants were routinely predarkened at 25 "C for 24 h prior to inoculation; groundnuts (cv. Natal Common) for a minimum of 48 h. All host range study inoculations were made by grinding young systemically infected soybean or cowpea tissue in 0.067 M phos- phate buffer, pH 7.7. Back tests from inoculated and from newly produced non- inoculated leaves were made after 28 days.

Sucrose density gradients were made by layering 4, 7, 7, and 7 ml of 10, 20, 30 and 40% sucrose in 0.01 M phosphate buffer, pH 7.7, respectively in 3 x I in centrifuge tubes ; 0.4 ml partially purified virus preparations were layered onto these and centri- fuged for 120 min at 24000 rev/min.

Antisera were prepared by injecting rabbits intravenously with I ml of a purified virus preparation, followed 7 days later by an intramuscular injection of I rnl virus emulsified with I ml Freund's incomplete adjuvant, and again 14 days later by a second, similar injection. Rabbits were bled 28 days after the final injection.

Serological tests were made in precipitin tubes (I ml each of antigen and antiserum, at various dilutions) held in a waterbath at 37 "C. Final records of reactions were made after 3 or 4 h.

Peanut mottle virus I73 Because of the likelihood of spontaneous, non-specific precipitation in saline-

antigen systems where preparation of partially purified antigen involves the use of chloroform (M. Hollings, personal communication), the following procedure was routinely used. Equal volumes of 0.3 M-NaCl were added to partially purified virus preparations ; these were incubated overnight at ambient laboratory temperatures (c. 20 "C), and centrifuged for 10 min at 75008 before initiation of serological tests. As an additional control, a range of antigen dilutions in normal saline were incubated at 37 "C.

RESULTS

The virus isolates Peanut mottle virus (PnMV) was isolated from groundnuts showing various

rosette and mottle symptoms, from stunted plants, and from apparently healthy plants: it could not be associated definitely with any specific symptom in the field. Characteristic PnMV symptoms on young plants are cupped leaves, prominent veins and depression of interveinal tissue; in mature plants, a mild or a dark green blotch mottle. Only rarely were these symptoms recorded in fields. In contrast, marked symptoms were invariably found in field-grown soya, but were variable and dependent on variety. Leaves were severely crinkled, blistered and malformed, or showed a mild or severe, yellow or green, mottle or mosaic.

Text-fig. I indicates the distribution of PnMV in East Africa.

Host range and symptoms Arachis hypogaea (cv. Natal Common). Isolates from groundnut and soybean in-

duced distinctive symptoms in cv. Natal Common under glasshouse conditions. The third or fourth leaflets (c. 14 days after inoculation) remained cupped inwards for several days. When these unfurled, main veins were prominent, with depression of interveinal tissue. As these leaflets assumed a dark green colour (which is normal with groundnut, where leaflets are light yellow-green on emergence), mottle symptoms developed. These varied in intensity between isolates, and ranged from a diffuse indistinct mottle to a pronounced dark green blotch mottle (Plate, fig. I).

Phaseolus vulgaris (cv. The Prince). Local chlorotic lesions developed in 8-10 days, became necrotic and subsequently spread dendritically along the veins (Plate, fig. 2).

Glycine max (cv. HLS 541). The first trifoliate leaves developed systemic yellow vein-clearing in 8-14 days; this disappeared and a dark green mottle developed. Sub- sequently produced leaves became progressively more severely crinkled, blistered and deformed (Plate, figs. 3,4). There was variation in severity of crinkling between isolates.

Vigna unguiculata (Mak I). Chlorotic local lesions developed at temperatures above 24 "C. An indistinct narrow dark-green vein-banding was induced 14-21 days after inoculation; subsequent leaves produced chlorotic spotting and light green mottling (Plate, fig. 6).

Cassia occidentalis. All isolates induced vein-clearing of some of the veins of the first leaflets, followed by a distinctive yellow mottle (Plate, fig. 5) .

In addition to these diagnostic hosts, the following species were susceptible : Systemic mottle symptoms were induced in Calopogonium mucunoides Desv.,

Cassia bicapsularis L., C . obtusifolia L., Cicer arietinum L., Macroptilium lathyoides

I74 K. R. BOCK (L.) Urb., Nicotiana clevelandii L., Pkum sativum L., Trifolium subterraneum L., Tr@nella foenum-graecum and Vigna oblongifolia var. parvzjlora (Bak.) Verd.

Symptomless systemic infection occurred in Lupinus angustifolius L., Mumoptilium atropurpueurn (DC.) Urb., Trifolium hybridium L. and Vigna oblongifolia A. Rich.

Insusceptible species included Cajanus cajan (L.) Millsp., Centrosema pubescens Benth., Chenopodium amaranticolor, C. quinoa, Citrullus Eanatus (Thumb.) Mansf.,

KENYA

TANZANIA

0"

5"

09 s

30. E 35' 40' Text-fig. I . Distribution of peanut mottle virus in East Africa. PnMV field hosts:

0, groundnut; 0, soya; A , cowpea; A, Voandzeia.

Peanut mottle virus I75 Crotalaria brevidens Benth., C. juncea L., C. pallida Ait., Cucumis sativus L., Cucurbita pep0 L., Gomphrena globosa L., Lens esculenta Moench., Nicotiana glutinosa L. and N. tabacum L.

Physical properties Dilution endpoint. Cowpea or soybean sap was infective at dilutions of 10-2, rarely

Thermal inactivation point. Most infectivity was lost when infective cowpea or soy-

Longevity in vitro. Expressed sap retained infectivity for 2, but not 3, days at 20 "C. Effect of freezing. Infectivity was only slightly reduced after freezing systemically

infected young cowpea leaves at - 12 "C for 12 wk. Effect of p H on infectivity. Sap was expressed from systemically infected leaves in

0.06 M phosphate buffer at pH 5 - 5 , 6.5, 7-5 and 8.0 (2 ml buffer/g leaf tissue) and infectivity of the extracts assayed on P. vulgaris. Number of lesions/leaf for the treat- ments were 10, 21, 122 and 150 respectively, indicating an intolerance of pH values below 7.0.

Effect of clarqying agents in infectivity. Fifty ml aliquots of infective sap extracted in 0.06 M phosphate buffer with 0.01 M EDTA and 0.1 % thioglycollic acid (pH 7-3) were each treated with different clarifying agents. The clarified preparations were centri- fuged at 78000g for 90 min; pellets were resuspended in 0-01 M phosphate buffer and the suspensions assayed for infectivity (Table I). Although preparations treated with chloroform were not as free of residual plant material as butanol or butanol/ chloroform preparations, the effect of the latter treatments on virus yield precluded their use.

Table I. EfSect of clarifying agent on infectivity

at IO-~, and not a t IO-*.

bean sap was heated for 10 min at 54 "C, and all was lost at 56 "C.

Infectivity (mean no.

Treatment lesions/leaf)

Ether (equal volumes) N-Butanol

7 ml/Ioo ml sap 8 ml/Ioo ml sap 9 ml/roo ml sap

Butanol/chloroform 1:1*

3 : 1

volumes)

2: I

Chloroform (equal

84.3

0.9 0.9 0.3

83.6 83.4 40.0

306.4

* Ratio of a mixture of equal volumes of butanol and chloroform to volume of sap.

PuriJication Satisfactory yields of virus were obtained only from infected cowpea cultured at

27 "C. A specific light scattering zone (19-21 mm below the meniscus) formed in sucrose density gradients only with preparations made from cowpea cultured at high temperatures; preparations from plants maintained in normally heated glasshouses at

176 K. R. BOCK EAAFRO, where temperatures were 21-23 "C, never yielded sufficient virus to form a light scattering zone.

Material was homogenized in 0-5 M sodium citrate buffer containing I % mercapto- ethanol ( I ml buffer/I g tissue), strained, stirred with an equal volume of chloroform at laboratory temperature for 20 min, and clarified by centrifugation for 20 min at 20000g. The virus was sedimented for gomin at Iooooog, and resuspended in 0.01 M phosphate buffer (c. one-eightieth of the sap volume) overnight. Such preparations when diluted 1/10 with phosphate buffer gave one narrow, intense light- scattering zone in gradients, indicating an adequate virus yield for immunization.

Serology of isolates Antiserum to one isolate was prepared and tested against purified preparations of the

homologous virus diluted 1\10; the antiserum had a precipitin dilution end-point of 118192. In subsequent serological tests, dilution of purified preparations were stan- dardised between 1 /10 and 1/20; precipitin reactions of higher antigen dilutions (1/40) took longer (2 h in contrast to 10 min) and had consistently lower end-points (1/1024,

in contrast to 1/8192).

Table 2. Serological tests with fourteen virus isolates from dzjferent localities (Text-Jig. I )

Field host

Groundnut I Groundnut 2 Groundnut 3 t Groundnut 4 Groundnut 5 Groundnut 6 Soya I

Soya 2 Soya 3 Soya 4 Cowpea I Voandzeia I Voandzeia 2 Voandzeia 3

Locality

Serere, E. Uganda Masindi, W. Uganda Ukiriguru, N.W. Tanzania Ukiriguru, N.W. Tanzania Msabaha, Kenya Coast Mtwapa, Kenya Coast Nkozi, S. Uganda Kasese, W. Uganda Kitale, N.W. Kenya Kakamega, W. Kenya Bukoba, N.W. Tanzania Ukiriguru, N.W. Tanzania Ukiriguru, N.W. Tanzania Ukiriguru, N.W. Yanzania

Precipitin end-point reciprocal

EA-PnMV USA-PnMV-M2 antiserum antiserum

I .A ,

2048 #

I 024 I 024 2048 4096 8192 8192 8192 2048 c 8192 8192 2048 2048 2048 4096 2048 2048 1024 8192 256 512 I 28 256 I 28

* Not tested. t Rosette-resistant selection.

Fourteen isolates were selected on the basis of field host and locality, bulked in cowpea, purified, and subjected to serological tests with East African and American PnMV antisera. All groundnut, soya and cowpea isolates, irrespective of geographic occurrence, reacted similarly, but the Voandzeiu subterranea isolates apparently differed serologically ; PnMV antiserum from America (USA-PnMV-M2) was similar to EA-PnMV antiserum in all respects (Table 2).

Purified virus preparations were also used to study the serological relationships of East African PnMV isolates with other viruses of the PVY legume group. Antisera prepared against bean common mosaic virus, bean yellow mosaic virus, cowpea

Peanut mottle virus I77 aphid-borne mosaic virus, soybean mosaic virus, clover yellow vein virus, potato virus Y, tobacco severe etch virus, celery mosaic virus, iris mosaic virus and sugarcane mosaic virus did not react with any isolates of East African PnMV.

Electron microscopy The average length of 19 PnMV flexuous filamentous particles in purified prepara-

tions suspended in 0.01 M phosphate and stained negatively in 2% potassium phos- photungstate was c. 754 nm. Modal length of 50 particles measured from quick leaf dip preparations was 7 4 ~ 5 0 nm.

Transmission Aphis craccivora Koch transmitted PnMV from groundnut to groundnut and

soybean in 5 min; infectivity was not retained after 2 h. In seed transmission tests with groundnut, PnMV-infected plants were grown to

maturity, and the subterranean pods left in situ in the pots. After a brief period of drying, the pots were again watered. Germination of the seeds and subsequent growth closely paralleled the growth of groundkeepers in the field. Rate of seed transmission in such plants was 20 % (6 out of 3 I) ; infected plants showed typical mild symptoms.

PnMV was not transmitted through seed of cowpea (0/335) or soybean (0/204); leaves of the apparently healthy seedlings derived from seed from infected plants were assayed to Phaseolus vulgaris and no virus was detected.

Susceptibility of soybean cultivars Kuhn, Sowell, Chalkley & Stubbs (1968) have given an indication of a general

pattern of susceptibility of groundnut cultivars to PnMV. In the present studies, the soybean cultivars b o y , Beeson, Calland, Culter, Hawk, and Wayne, two F8 Mexico 13 x Acadian, one F 8 Mexico 13 x Mandarin, and twelve F 8 Mexico 13 x Pelican breeding lines, were tested for resistance to PnMV. Five seedlings of each cultivar or line were inoculated with a soya isolate in each of two separate tests. Every plant became infected with PnMV; although symptoms were not as severe as in the standard test variety, no indication of resistance was seen.

D I S C U S S I O N

PnMV has been identified in East Africa on the basis of morphology, serology, host range and reaction, transmission and physical properties. PnMV has been reported from North America (Kuhn, 1965) and from Venezuela (Herold & Munz, 1969); results of extensive surveys reported here indicate an extremely wide occurrence throughout Kenya, Uganda and Tanzania.

Kuhn (1965) and Herold & Munz (1969) suggested that PnMV might be similar to the mottle virus described by Storey & Ryland (1957), on the basis of similar symp- toms in groundnut, mechanical transmission and transmission by A. craccivora. However, PnMV differs markedly from both of Storey & Ryland’s (1957) two ground- nut mottle viruses. A virus inducing a mottle in groundnut similar to their Ngomeni mottle, and also isolated only from East African coastal regions, is at present under study. This virus, unlike PnMV, has stiffish 650 nm particles, occurs in relatively high

178 K. R. BOCK concentration in groundnut and soya, and causes a severe systemic reaction in Phaseolus aulgaris. Nor can PnMV be related to Storey & Ryland's (1957) Serere mottle; PnMV was only randomly associated with plants showing Serere mottle virus symptoms in the type locality, and is not associated with mottle symptoms in coastal areas, where the upcountry mottle virus apparently does not occur. All other reports of groundnut viruses in Africa are too brief or superficial for adequate comparisons to be made.

There is at present no evidence for the existence of distinctive groundnut or soy- bean strains of PnMV in East Africa such as have been described from America (Paguio & Kuhn, 1971 ; Sun & Hebert, 1971). Although East African isolates induce symptoms in groundnut which range from a diffuse, indistinct mottle to a pronounced dark green mottle, variation appears to be within these relatively close limits. The East African groundnut and soybean isolates appear remarkably uniform in physical properties, and differ from the type virus in thermal inactivation point (56 "C, as opposed to about 64 "C for American isolates) and longevity in vitro (48, as opposed to 24 h).

PnMV is widespread in soybean in East Africa; field soybean infection has been recorded recently in the United States (Kuhn, Demski & Harris, 1972) also, where it is suggested that the virus should be considered in soybean production and research programmes.

The virus isolated from Voandzeia subterranea is serologically related to PnMV, but appears, on present evidence, to be a distinct virus. In contrast to American and East African strains of PnMV which have been studied, the virus infects Cajanus cajan systemically, and Chenopodium arnaranticolor, C. quinoa and Gomphrena globosa locally; infection in groundnut is symptomless.

Results of experiments where conditions pertaining to field groundkeepers were simulated indicate that seed-transmission may be an efficient method of carry-over between dry seasons in Africa; the susceptibility of several Cassia species, both in East Africa in the glasshouse and in the United States in the field (Kuhn, 1965), also indicates the presence of permanent potential reservoirs of infection.

Kuhn (1965) and Sun & Hebert (1971) have demonstrated yield reductions of 25 %, 50% and 70% in experimental PnMV-infected groundnut varieties; the poten- tial effect on yield in the field is certainly great. Unfortunately, Kuhn et al. (1968) were unable to find immunity among 465 groundnut accessions from Africa, South America, Japan, India and Australia, although there was evidence of differences in susceptibility. The same problem occurs with soybean; we have found no evidence of resistance in a comparatively wide range of germplasm.

In several areas of Africa, breeding and selection for resistance to groundnut rosette disease has been moderately successful: unlike PnMV, rosette is spectacular in its symptom expression, and it is thus comparatively easy to score selections for resistance. American records of the yield losses caused by PnMV, together with the frequency of occurrence of the virus in East Africa, suggest that current and future groundnut and soybean improvement programmes must take into account resistance to PnMV. This will involve both the routine testing of improved varieties for susceptibility to PnMV, and evaluation of the effect of PnMV on yield.

dnnals of Applied Biology, 1-01. 74, h'o. 2

Peanut mottle virus I79 I thank Dr C. W. Kuhn, Dr J. P. Ross and Dr M. Hollings for gifts of antisera;

Mr R. D. Woods for electron microscopy; and Miss E. Ngugi and Mrs L. Magua for technical assistance.

This paper is published by permission of the Director, EAAFRO.

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HAYES, T. R. (1932). Groundnut rosette disease in the Gambia. Tropical Agriculture, Trinidad 9,211.

HEROLD, F. & MUNZ, K. (1969). Peanut mottle virus. Phytopathology 59, 663. KLESSER, P. J. (1965). Groundnut variety trials for resistance to virus diseases. Technical Com-

KUHN, C. W. (1965). Symptomatology, host range and effect on yield of a seed-transmitted

KUHN, C. W., DEMSKI, J. W. & HARRIS, H. B. (1972). Peanut mottle virus in soybeans. Plant

KUHN, C. W., SOWELL, G., CHALKLEY, J. H. &STUBBS, H. F. (1968). Screening for immunity to

PAGUIO, 0. R. & KUHN, C. W. (1971). A necrosis strain of peanut mottle virus. Georgia Aca-

STOREY, H. H. & RYLAND, A. K. (1957). Viruses causing rosette and other diseases in ground-

SUN, M. K. C. & HEBERT, T. T. (1971). Purification and characterisation of a severe strain of

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EXPLANATION O F PLATE

Fig. I. Systemic symptoms of PnMV in groundnut (cv. Natal Common). Fig. 2. PnMV local necrotic lesions in Phaseolus vulgaris (cv. The Prince). Fig. 3. Effect of PnMV on growth of the highly susceptible soybean cultivar HLS 541. Fig. 4. Systemically infected soybean leaf (cv. HLS 541). Fig. 5. Systemic mild mottle symptoms induced by PnMV in Cassia occidentalis. Fig. 6. Systemic chlorotic mottle caused by PnMV in cowpea (CV. Mak I).