plant pathology (1995) 44, 527-546

Plant Pathology (1995) 44, 527-546

Variation for pathogenicity among isolates of bean commonmosaic virus in Africa and a reinterpretation of the geneticrelationship between cultivars of Phaseolus vulgaris andpathotypes of BCMV

N.J. SPENCE and D.G.A. WALKEYHorticulture Research International, Wellesbourne, Warwick CV35 9EF, UK

Bean common mosaic virus (BCMV) isolates were collected from crops of Phaseolus vulgaris (bean)and from wild legume species in 13 African countries. Isolates of pathotype Via from both beans andwild legume species were predominant in central, eastern and southern Africa. Isolates of pathotypes I,III, IVa, IVb and Va were also found. Some isolates did not conform to previously publishedpathotypes, and therefore represent records of novel pathotypes. The susceptibility of various wildlegume species to BCMV was investigated and isolates of the virus obtained from Crotalaria incana,Rhynchosia sp., Macroptilium atropurpureum and Cassia occidentalis (synonym Senna occidentalis) wereaphid-transmitted both from P. vulgaris to their original host species and to P. vulgaris. Isolates ofBCMV from wild legume species were seed-transmitted in bean and in several other legume species. Thenatural occurrence of BCMV in wild legume species in Africa is probably a significant factor in theecology and epidemiology of the virus and possibly the evolution of isolates of the 'A' serotype whichinduce necrotic reactions in cultivars carrying the / gene for resistance. The occurrence of viruses otherthan BCMV from P. vulgaris and other legume hosts is also reported. The gene-for-gene modeldescribed by Drijfhout (1978) is reinterpreted to explain the variation for pathogenicity, and it isproposed that there may be genes which control the temperature sensitivity of necrosis in combinationwith the / gene.

INTRODUCTION

Bean common mosaic potyvirus (BCMV) has aworldwide distribution and is economicallyimportant throughout Africa, Asia, Europe andNorth, Central and South America. The virus cancause severe crop losses, and its severity is usuallydependent on the incidence of aphid vectors(Morales & Bos, 1987). BCMV is the mostimportant virus disease of Phaseolus vulgaris(beans) in Africa. However, other viruses havebeen isolated from this host species, includingbean yellow mosaic potyvirus (BYMV), cucumbermosaic cucumovirus (CMV), peanut mottlepotyvirus (PeMoV), cowpea mild mottle carla-virus (CMMV) and blackeye cowpea mosaicpotyvirus (BICMV) (Vctten & Allen, 1991).

Natural hosts of BCMV are mainly restrictedto Phaseolus spp., especially P. vulgaris (Drijfh-out, 1978). However, BCMV has been isolatedfrom other leguminous hosts, including Vignaunguictitata (Zaumeyer & Thomas, 19S7), Vigna

radiata (Kaiser & Mossahebi, 1974), Crotalariajuncea (Singh & Singh, 1977), Crotalaria striata(Sarkar & Kulshreshtha, 1978), Rhynchosiaminima (Meiners et ai, 1978) and Lupinusluteus (Frencel & Pospieszny, 1979).

BCMV causes two types of symptom in P.vulgaris: common mosaic (Fig. 1), which mayseverely reduce yield, and systemic necrosis('black root") (Fig. 2), which can cause plantdeath. Black root develops as a consequence of ahypersensitive reaction to so-called necroticstrains of BCMV, but only in cultivars posses-sing the dominant /gene which confers resistanceto mosaic-inducing strains (Drijfhout, 1978).Necrotic strains were first identified in Europe(Hubbeling, 1963; Drijftiout & Bos, 1977), andBCMV epidemics caused by necrotic strains havesince been reported in the USA (Kelly el ai,1984; Provvidenti et ai, 1984; Myers et ai. 1990).Recent surveys in Africa suggest that necroticstrains predominate over much of eastern andsouthern Africa, causing severe economic losses

\ . J. Spence & D. G. A. Walkey

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vuUiv.ir Ldrrvinii the / iicne k:4u>ed bv bean ijommon

used to reinterpret the gene-tor-gene modeldescribed b\ Driilliout ( I'^'Sl

M M F R I M S AND METHODS

Sur>e\s

f leld sur\e\s were earned out in Cc llaboratic^nwith personnel Irom Cenlro Internaeional deVgriciillLira Tropical (CI-XTl and National BeanProgrammes in Lesotho. Malawi, Swaziland.I gand,i and Zimbabwe (Januarx 19'JO), Ethio-pia iSeptember I' ' Oi. Morocco (No\emherI'l XH. Rwanda, Tau/aaia, Luanda and Zaire(Ma\ I'J'^ll. Burundi. K.en>a and RwandaiNovember \^>'->\\ In ,iddition. >aniples werereceixed al W ellesbourne direetlx Irom .\tnca

Host plants, \irus isitlatcs, inoculations andmuintenanee of eultures

Seeds ol test pl,inb were germinaievl in plasticboxes iMi d,imp cellulose wadding h\ inculxuins;(liein lor 4 d,i\s ,it 2,' C The gerniiiiaied seedswc'ie plaiued m M2 I eviiictou compost ^hisot\s,I K,) in •'-ciii plasiic pois Plants were grown inan iiiseci-proiil iiKisshouseat 2^ (.\ from tX'tober

Variation for pathogenicity among isolates of BCM V 529

Table I. Differentiation and grouping of bean common mosaic virus pathotypes (Drijfhout, 1978)

Pathotype of the virus and representative strains

Differential hosts

INLlUSl

IINL7

IIINL8

IVaUS5FLANVRS

Pathogenicity genes

PQ POP\

PO

P2

POP\P]'

IVbNL6US3US4

POP\PI'

VaUS2NY15

POP\

P2

VbNL2

POP\

P2

ViaNL3

POP\PI'P2

VIbNL5

POP\PI'P2

VIINL4US6

POP\P\'

Group Cultivar P2'

1 Double WhiteThe Prince +" + + + + + + - I - - I - +Striagless GreenRefugee (SGR)Common Red Mexican (CRM)

2 Redlands Greenleaf C (RGC)Puregold Wax (PGW) - ' ' +t ' - -I- + -t-t -i- -l-t + -I-Imuna

3 Redlands Greenleaf B (RGB)GN UI 59 (GN59) - - - + - l - - - - l - t + t - l -GN UI 123 (GN123)

4 MicheliteSanilac — - + - - + + + + -Pinto 111 (PI 11)Red Mexican 34 (RM34)

5 Pinto 114 (PI 14) _ _ _ _ _ + + + + _

6 M o n r o eRed Mexican 35 (RM35) - - - _ _ _ _ _ _ +G N U I 3 1 ( G N 3 1 )

7 I V T 7 2 1 4 _ _ _ _ _ _ _ _ _ _

8 Black Tur t le Soup (BTS) - - N " - n ' - - N N -Widusa

9a Jubila _ _ _ _ N _ n N N -

9b Improved Tendergreen ( ITG)T o p c r o p ( T C ) _ _ _ _ n - n N N

10 Amanda _ _ _ _ _ _ _ _ N -

11 IVT7233 _ _ _ - _ _ _ _ _ _

*-l-, host group susceptible to systemic infection.^ - , host group resistant to systemic infection.'^+t, symptomless host but systemic infection detected by ELISA.••N, systemic necrosis at 26 C and 32 C.'n, systemic necrosis at 32°C, but not at 26°C.

to March supplementary lighting was provided Infected plant material was collected as leaves,to give a daylength of 16 h. Seeds of the genera If samples other than bean were collected,Chenopodium and Nieotiana were sown directly additional herbarium material was preserved inin sifted peat compost and germinated seedlings a flower press and photographs were taken to aidwere pricked out into individual pots. identification. For each sample, records were

530 N. J. Spence & D. G. A. Walkey

Table 2. Standard viruses and virus strains used

Virus strain

BCMV NLlBCMV NL3BCMV NL4BCMV NL5BCMV NL6BCMV NL8BCMV NVRSBCMV NY 15BICMV NRCABMVPeMoVAMV

Source

E. DrijfhoutE. DrijfhoutE. DrijfhoutH.J. VettenE. DrijfhoutE. DrijfhoutD.G.A.WalkeyH.J. VettenH.J. VettenH.J. VettenH.J. VettenD.G.A.Walkey

Propagationhost

P. vulgari^P. vulgarisP. vulgarisP. vulgarisP. vulgarisP. vulgarisP. vulgarisP. vulgarisN. clevelandiiN. clevelandiiN. clevelandiiN. clevelandii

Country oforigin

The NetherlandsThe NetherlandsThe NetherlandsGermanyThe NetherlandsThe NetherlandsUKGermanyGermanyGermanyGermanyYemen

' cv. Double White.

taken of location, altitude, host species, cultivar(if known) and disease severity. Leaf sampleswere placed between two sheets of filter paper(Whatman No.l), lightly moistened with waterand placed in a polythene bag. The samples werestored in cool bags in the field and laterrefrigerated at 4-5 'C until they reached thelaboratory (usually within 2 3 weeks). Addi-tional similar leaf samples were immediatelydried in plastic scintillation vials containingapproximately 10 g of coarse CaClj.

Samples (fresh or dried) were homogenized in1 % K2HPO4 solution containing 0-1

g/ml) and used for sap transmission to appro-priate test seedlings, including P. vutgaris cv.Double White (Dubbele Witte), Chenopodiumquinoa, Nicotiana benthamiana, N. clevetandii,Vigna unguiculata and V. radiata. Sap inoculumfor sub-culturing was prepared in the same way.Some virus isolates were obtained from infectedseedlings grown from seed samples collected inAfrica.

BCMV strains representing pathotypes I(NLl), III (NL8), IVa (NVRS), IVb (NL6), Va(NY 15), Via (NL3), VIb (NL5) and VII (NL4)(Table I) were maintained in bean cv. Double

Table 3. Antibodies used in ELISA

Name Type Source

BCMV NLlBCMV NL3BCMV NL4BCMV NL5BCMV NL6BCMV NL8BCMV NY 15BCMV NVRSBICMV-NRCABMVPeMoVbc-I-3bc-l-IA412197

PabPabPabPabPabPabPabPabPabPabPabMabMabMabMab

NJ . Spence, HRI WellesboumcN.J. Spence, HRI WellesboumeNJ . Spence, HRI WellesboumeH.J Vetten, GennanyN J . Spence, HRI WellesboumeN J . Spence, HRI WellesboumeH.J. Vetten, GermanyD G A . Walkey, HRI WellesboumeH.J. Vetten, GennanyH J. Vetten, GemianyH.J. Vetten, GermanyH.J. Vetten, GermanyH.J. Vetten, GemianyG I Mink, Washington, USAG I Mink, Washington, USA

Pab, polycloi\«l antiscrum, Mab, monoclonal antibody.

Variation for pathogenicity among isolates of BCMV 531

Table 4. Number of isolates of bean common mosaic virus derived from Phaseoius vulgaris samples only fromcountries in Africa and assigned to particular pathotypes

Country

BurundiEthiopiaKenyaLesothoMalawiRwandaSwazilandTanzaniaUgandaZaireZambiaZimbabwe

Total

Number ofsamplescollected

5810369314278365843251452

609

Approximate percentageof each pathotype

I

110000011000

4

3

11

000000000000

0

0

III

800004020000

14

9

IVa

000000000001

1

1

IVb

210012102012

12

8

BCMV pathotypes

Va

001000000000

1

1

Vb

000000000000

0

0

via

100727

2216

11148

79

53

VIb

000000000000

0

0

VII

000000000000

0

0

Novel

537163160312

38

25

Total

265

153

1431

3151446

13

149

100

Approximatepercentage

of samplesinfected

455

22103340

826322

4325

White and are referred to as 'standard' strains tobe used for comparison with unidentifiedisolates. Cultures of BICMV, cowpea aphid-borne mosaic potyvirus (CABMV), PeMoV andalfalfa mosaic virus (AMV) were also maintainedfor comparison (Table 2).

Diiferential bean cultivars

Original seed was obtained from E. Drijfhout(Agricultural Research University, Wageningen,The Netherlands) and multiplied at Welles-bourne in insect-proof polythene tunnels. Up to22 cultivars were used to characterize BCMVisolates on the basis of systemic infection(Drijlhout, 1978). A minimum of two cultivarsper host resistance group were used in each test,and four seedlings of each cultivar wereinoculated as soon as the primary leavesunfolded. In addition, two further plants ofcultivars in resistance groups 8, 9a, 9b and 10(which possess the dominant / gene) wereinoculated with each isolate and maintained ina controlled environment cabinet at 32°C inorder to identify those isolates which inducedsystemic necrosis only at this higher temperature.

Each plant was scored for symptoms atintervals up to 4 weeks after inoculation,diflerential cultivars that developed question-able symptoms were back-inoculated to plants of

cv. Double White to check for virus infection, orthe suspect plants were tested for infection byELISA. Potentially mixed infections were back-inoculated from individual cultivars to a new setof differential cultivars. Isolates were assigned topathotypes according to the reaction patterns ofthe differential host cultivars. The reactionpattern on the differential host cultivars follow-ing inoculation with the standard BCMV strainswas investigated in order to confirm the originaldescriptions by Drijfhout (1978).

Enzyme-linked immunosorbent assay (ELISA)

A number of monoclonal antibodies and poly-clonal antisera (Table 3) were used in ELISA toaid the identification of the BCMV isolates andother viruses (Clark & Adams, 1977). Mono-clonal antibodies bc-1-3 (BC3) and 197 werebroad-spectrum, reacting in plate-trapped anti-gen ELISA (PTA-ELISA) with all BCMV strainsand other potyviruses including BICMV,CABMV and PeMoV. In contrast, monoclonalantibodies bc-l-lA4 (BCl) and 12 were highlyspecific to BCMV strains of the 'A' serotype only.Each survey sample was tested with thesemonoclonal antibodies by PTA-ELISA in dupli-cate wells. To aid identification, samples were alsotested in direct double-antibody sandwich ELISA(DAS-ELISA) with a number of polyclonal

532 N. J. Spence &D.G. A. Walkey

Panwyp.

Fig. 3. Distribution of pathotypes of bean commonmosiac virus obtained from Phaseolus vutgaris growingin Africa

antisera raised to specific BCMV strains andother potyviruses.

Aphid transmission

Myzus persicae was used for aphid transmissionexperiments. Aphids were reared on healthyplants of Brassica juncea cv. TendergreenMustard and starved for 2h before an accessfeeding period of c 5min on infected leaves. Tenor more aphids were then removed with apaintbrush, placed on healthy seedlings andkilled 24 h later by spraying with heptenophos(Hostaquick).

Host range and seed transmission

Seeds of 29 wild legume species were tested forsusceptibility to BCMV. Seeds were soaked indistilled water for 24 h before being nicked with ascalpel blade to aid germination; seedlingproduction was as described for beans. Fiveseedlings of each species were sap-inoculatedwith the following standard BCMV strains: NLl,NL3, NL4, NL6 and NL8, and isolate 30 fromCrotalaria incana L. Symptoms were recordedweekly for 4 weeks, ut which time all seedlingswere tested by HLISA for systemic infection.

Plants of the 29 wild legume species which hadbeen inoculated with BCMV isolates wererepotted into l8-cm pots 8 weeks after inocula-tion. Flowering was induced by growing theplants under short-day conditions (8-h photo-period). Plants were either allowed to self-pollinate or were hand-pollinated with a paint-brush. The resultant seeds were harvested andsubsequently germinated. Four weeks aftersowing, virus symptoms were recorded and leafsamples from every plant were tested by ELISAfor the presence of virus.

The seed transmission of virus isolates fromwild legumes in P. vulgaris was tested byinoculating four plants of the bean cvs DoubleWhite and/or Sutter Pink with each isolate. Atflowering, c. 3 weeks after sowing, the plantswere repotted into 18-cm pots. Plants wereallowed to self-pollinate and the restiltant seedswere collected and subsequently tested for seed-transmitted virus by visually recording virussymptoms in germinated seedlings, and bytesting for the presence of virus by ELISA.

RESULTS

BCMV isolates from P. vulgaris

In general, the reaction patterns observedfollowing inoculation of the differential beancultivars with the standard stnuns of BCMVwere consistent with those described by Drijfh-out (1978). The small differences that wereobserved can probably be attributed to environ-mental variation (particularly temperature).

BCMV was isolated from 149 of the 609samples of P. vulgaris collected in Africa (Table4). From the remaining samples either no viruswas detected, or BCMV was detected by ELISAbut was not isolated. Viruses other than BCMVwere isolated from some samples or detected byELISA, but not isolated (Spence & Walkey,1994). The variation in the reaction patternsobserved on dilTerential cultivars followinginoculation with the BCMV isolates fromAfrica was considerable, but the isolates havebeen assigned, wherever possible, to the patho-type to which their reaction patterns most doselyconformed. In some cases, assignment to apathotype was clear<ut, as reaction patternswere identical to those of the standard strainrepresentative of the pathotype In other casesthere were small but distinct and consistentdifferences between the reaction phenot)peobserved and that of standard strains. This


Table 5. The reactions of differential host cultivars to isolates of bean common mosaic virus expressing novelpathogenicity phenotypes compared with NL3

Diiferential hosts

Group

NL3Cultivar

100S

874B

191Z

3515Za

3507Za

161M M

9a

9b

10

The PrinceSGRDouble WhiteSutter PinkCRM

PGWRGC

RGB

MicheliteSanilac

Pinto 114

MonroeRM35GN31

BTSWidusa

Jubila

ITGTC

Amanda

Pathogenicity genes

-t-++

nfntnt-I-

_ b

-l-t'

-t-

nt-t-

nt

- I -ntntnt-I-

nt

-1--1--1-nt

+ntntnt

-l-ntntnt

++-

-

N

N

NN

-

POPIPl^P2

++

-

nt

NN

Nn

a

POPI

P2

+

nt

-

NN

nNN

-

POPI

P2

-1-

+-

nt

NN

n

NN

-

POPI

P2

-1-

-1-

-

-

NN

.1

NN

-

POPI

P2

+-

-

-

n'n

n

NN

-

PO

P2

++

-

nt

N»

1

-

-

POPIPl-P:

++

-

nt

NN

n

-

POPIPIP2

'+, host susceptible to systemic infection,''-, host resistant to systemic infection,'+t, symptomless host but systemic infection detected by ELISA,••N, systemic necrosis at 26°C and 32°C.'n, systemic necrosis only at 32°C," nt, not tested.Origin of isolates: B, Burundi; M, Malawi; S, Swaziland; Z, Zimbabwe; Za, Zaire,

variation included the failure of an isolate toinfect one particular cultivar from a particularhost resistance group even though other cultivarsof the same group were infected. Isolates whosereaction phenotype deviated from that of thestandard strain by variations such as this werestill assigned to the pathotype they most closelyresembled. Isolates whose reaction patterns didnot conform to any previously described patho-type were considered to represent novel patho-types.

Approximately 53% of BCMV isolates fromP. vulgaris were assigned to pathotype Via, thereaction phenotypes on differential bean culti-vars being similar to NL3, Isolates of pathotypeVia occurred widely in all the areas surveyedexcept Ethiopia, Isolates assigned to pathotypeIII. similar to NL8, had a more limiteddistribution, being found only in Burundi,Rwanda and Tanzania, Isolates assigned topathotype I, similar to NLl, were toundoccasionally in Burundi, Ethiopia, Tanzania

534

I S +

AT. J. Spence & D. G. A. Walkey

l l + + l l l l e a l l

I I + + I l l l e e l I

I I + + I I l l c c i I

S

+ C C C + I I I I I I

i

I II

SI

SI

SI

m W + C + + C I I + I I i c I l Z Z l c l

+ C I I + I I I c I l Z Z i I I

+ C + + I I I i c l l Z Z i I I

+ c c + + + + + + c l c Z Z I I I I

z

+ C + + C + I l + + l c l l Z Z c l l i S a , SI

+ C + + C + I i + + i c i i z z i I I i S s ; SI

l c + + = I + + + I I C Z Z 1 1 1 1 S 5 ; SI

+ C + C C + + I + + I I I c Z Z I I I I £ 5 1 SI

+ + + c s I I + + + + I i c z z I I I I £ 5 ; 5 ; S I

+ "c+ + c I + + + + I H I I Z Z C Z Z I £ S i ' s ; SI

+ + + + + I I I + + I i i c z z i I I i S SI

+ + + + + + + + + + + I I l i z y y y I P r ' r g*

13

I

•s

I"o

•ac

l


Table 7. The reactions of differential host cultivars to isolates of bean common mosaic virus expressing novelpathogetiicity phenotypes compared with NL6

Differential hosts

Group Cultivar

1

2

3

4

5

6

8

9a

9b

10

The PrinceSGRDouble WhiteSutter PinkCRM

PGWRGC

RGBGN59

MicheliteSanilac

Pinto 114

MonroeRM35GN31

BTSWidusa

Jubila

ITGTC

Amanda

Pathogenicity genes

Nil t\

+ '-(-H-+-1-

H--t-

+_-

-_-nt

n"n

n

nr

-

POPI

1771^ /

z+nt'ntnt+_ b

-

__

-

-

-

-

nt

N'N

n

nn

-

PO

4mT

-1-ntH--1-nt

_-

nt

_-

-

_-nt

nn

-

nn

-

PO

404T

+

-f

nt+_-

-

-

-

--

nn

n

nn

-

PO

887oo /B

+nt

+nt

_-

nt

-

-

nt--

nn

-

nn

-

PO

I4Q117

M

-1-ntntnt-1-

_

-

_

-

-

-

-

nt

nn

n

nn

-

PO

I7SI / O

M

-1-ntntnt-t-

_-

-

_

-

-

-nt

nn

n

nn

-

PO

407tu /T

-1-ntH--(-nt

-1-+

nt

_

-

-

-nt

NN

-

n-

-

POPI

1008Zm

nt-1-

+nt

+

nt

_

-

-

nt_-

nn

-

n.1

-

POPI

1701/7

M

-1-ntntnt+

-t-t'

_

-

-

-

-nt

NN

-

nn

-

POPI

1 ^1

M

-1-ntntnt+

4-

_

nt

-

-

_-nt

Nn

n

NN

-

POPI

QC-1

E

-1-nt-1-+nt

_

_nt

-

+

nt--

nn

n

nn

-

POPI

P2

'+, host susceptible to systemic infection,""-, host resistant to systemic infection,'N, systemic necrosis at 26-C and 32°C.•"n, systemic necrosis at 32 C but not at 26 C.'+t, «ymptomless host but vims detected by ELISA,' nt, not tested.Origin of isolates: B, Burundi; M, Malawi; R, Rwanda; T, Tanzania; Z, Zimbabwe; Zm, Zambia,

and Uganda, and an isolate assigned to patho-type IVa, similar to US5, was found in only onesample from Zimbabwe. The distribution ofisolates assigned to pathotype IVb, similar toNL6, was more widespread and they werefrequently isolated from infected seeds. The(ingle isolate in the survey which was assignedto pathotype Va, similar to NY IS, was found inwestern Kenya, whilst all other isolates obtainedin Kenya were assigned to pathotype Via (Fig,3), Isolates with apparently novel pathotypes

were found infecting P. vulgaris in all of thecountries surveyed except Uganda, The details ofthe pathogenicity phenotype of some of theseisolates are given in Tables 5-8, together with anindication of the pathogenicity genes of theisolate.

In Ethiopia there was a much lower incidenceof BCMV than in the other areas surveyed, andthe absence of isolates of the 'A' serotype issignificant. When infection by BCMV was found,the symptoms were generally mild. Only two

536 N. J. Spence & D. G. A. Walkey

Table 8, The reactions of differential host cultivars to isolates of bean common mosaic vims exprewing novelpathogenicity phenotypes compared with NY 15

Differential hosts

Group

1

2

3

4

5

6

8

9a

9b

10

Cultivar

The PrinceDouble WhiteSutter Pink

PGWRGC

RGB

MicheliteSanilac

Pinto 114

MonroeRM35GN31

BTSWidusa

Jubila

ITGTC

Amanda

Pathogenicity genes

NY15

-

+

nt

_

-

-

POPI

P2

961K

+

+ -1

-

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970K

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"+, host susceptible to systemic infection.""-, host resistant to systemic infection.'-)-t. symptomless host but systemic infection detected by ELISA, tolerant reaction.••nt, not tested.Origin of isolates: K. Kenya; B, Burundi. Isolate 963 was obtained from Crotalaria comartestiana\ all other isolateswere obtained from P. vulgaris.

isolates of BCMV were obtained which wereboth of the 'B' serotype, and these were assignedto pathotypes I and IVb. In addition, threeisolates (277,286 and 319) of an apparently novelpathotype were found at dilTerent locations in theRift Valley; these were confirmed to be of the 'B'serotype of BCMV (Table 6).

In most countries there was little differencebetween the strains found at or near researchstations and those found in farmers' fields. InMalawi, however, isolates assigned to pathotypeVia were only found near the Bunda College andMakapwa Research Stations, while isolatesassigned to pathotype IVb were only found infanners' fields. The recovery of a single isolate

conforming to pathotype IVa from the HarareResearch Station, Zimbabwe, may have origi-nated from imported virus-infected seed, becauseno other isolates of this type were found inZimbabwe.

The 'A' serotype of BCMV was detected byELISA in several bean plants collected during avery limited survey in Morocco. Howevtr,BCMV was always present in a mixed infectionwith alfalfa mosaic virus (AMV). and as a resuHthe reaction of differential bean cultivars to theseisolates was not determined.

Full details of all BCMV isolations madeduring the survey htve been documentad bySpenoe& Walkey (1994),


Table 9. Bean common mosaic virus isolates obtained from wild legume species inAfrica

Isolatenumber

283038145465499531820830836956963

Host species

Crotataria incanaCrotalaria incanaGlycine maxRhynchosia sp.Cassia hirsutaMacroptilium atropurpureumVigna unguiculataCassia sopheraCassia sopheraCassia sopheraVigna vexillataCrotataria comanestiana

Country oforigin

UgandaUgandaUgandaMalawiUgandaUgandaRwandaRwandaRwandaRwandaKenyaKenya

BCMVserotype"

AAABBAAAAABB

BCMVpathotype

NovelNovelNovelIVbIViaViaNovelNovelNovelVaNovel

° Serotype determined using monoclonal antibodies 197 and 12.

BCMV isolates from wild legume species

BCMV was isolated from several wild legumespecies in Kenya, Malawi. Rwanda and Uganda(Table 9), Isolates 28 and 30 from Crotalariaincana and isolate 38 from Glycine max obtainedfrom Nakabango, Uganda were all of the 'A'serotype and produced a similar set of reactionpatterns on differential host cultivars. Systemicveinal necrosis was observed in cultivars of hostresistance groups 1 and 4, but this reaction wasnot the typical hypersensitive reaction associatedwith the action of the / gene. The phenotypicreaction pattern was not similar to any pre-viously described BCMV pathotype, and theisolates were therefore considered to represent anovel pathotype (Table 5), Isolate 465 fromCassia hirsuta (synonym Senna hirsuta) obtainedat Bukalasa, Uganda was ofthe 'B' serotype. andfollowing inoculation of differential host cultivarsthe reaction pattern conformed to pathotype I,An isolate of the 'A' serotype (499) from Macro-ptilium atropurpureum obtained at Mubuku,Uganda conformed to pathotype Via followinginoculation of differential bean cultivars.

An isolate ofthe 'A' serotype (531) from Vignaunguiculata growing as a weed in a bean plot inRwanda conformed to pathotype Via, whilethree other isolates of the 'A' serotype (820, 830and 836) from Cassia sophera (synonym Sennasophera) obtained from different sites in Rwandadisplayed a novel set of reactions patterns ondifferential cultivars, und were considered to bepreviously undescribed pathotypes (Table 6),

Isolate 145 from a Rhynchosia sp, (not inflower) growing at Champhira, Malawi was ofthe 'B" serotype. This isolate conformed topathotype IVb following inoculation of differ-ential bean cultivars.

At Nyangusu, near Kisii, Kenya an isolate(956) of the 'B' serotype was obtained fromVigna vexillata, and the reaction pattern ondifferential host cultivars assigned it to pathotypeVa, At Nyangena. c, 30 km from the Nyangususite, an isolate (963) ofthe 'B' serotype obtainedfrom Crotalaria comanestiana yielded a novelreaction pattern and was therefore considered torepresent a novel pathotype (Table 8),

The survey indicated that herbaceous wildlegume species were numerous in Burundi.Malawi, Rwanda. Uganda and the wetter partsof Ethiopia and Kenya, Fewer herbaceouslegumes were observed in other areas, and nosamples were collected from wild legumes inLesotho. Swaziland. Zaire or Zimbabwe, as nonewere observed to have virus symptoms.

Full details of all BCMV isolations made fromwild legumes in the different countries have beendocumented by Spence & Walkey (1994),

Isolation of viruses other than BCMV

BCMV was the virus most commonly isolatedfrom P. vulgaris in Africa, but PeMoV and AM Vwere also found infecting P. vulgaris, and CMVwas isolated from P. lunatus in Ethiopia, PeMoVwas also isolated from wild legume speciescollected in bean-growing areas. Several isolates

538 N. J. Spence &D.G.A. Walkey

Table 10. Summary of viruses other than bean common mosaic virus isolated from legume hostsin Africa

Isolate

31225228242250313314316336338344347348366428429430434435522945

* Tentative

Country

UgandaEthiopiaEthiopiaEthiopiaEthiopiaEthiopiaEthiopiaEthiopiaEthiopiaEthiopiaMoroccoMoroccoMoroccoMoroccoTanzaniaTanzaniaTanzaniaTanzaniaTanzaniaRwandaKenya

name.

Location

NakabangoJimaJimaBongaDidLake ShallaLake ShallaNazrethMelkassaMelkassaBeni MellalBeni MellalBeni MellalBeni MellalMoshiMoshiMoshiMoshiMasasaniMusambiraMeru

Host species

Arachis hypogeaCassia sopheraCassia sopheraCassia sopheraPhaseotus lunatusPhaseotus vulgarisPhaseolus vulgarisCassia occidentalisCassia laburnifoliumCassia laburnifoliumPhaseolus vulgarisPhaseolus vulgarisPhaseolus vulgarisPhaseolus vulgarisPhaseolus vulgarisPhaseolus vulgarisPhaseotus vulgarisPhaseotus vulgarisPhaseolus vutgarisVigna sp.Cassia sp.

Virus isolated

Peanut mottle potyvinisPeanut mottle potyvimsPeanut mottle potyvirusPeanut mottle potyvimsCucumber mosaic cucumovinisPeanut mottle potyvimsPeanut mottle potyvimsCassia severe mosaic potyvims'Unidentified potyvimsUnidentified potyvims (as 336)Alfalfa mosaic vimsAlfalfa mosaic vimsAlfalfa mosaic vimsAlfalfa mosaic vimsUnidentified potyvirusUnidentified potyvimsUnidentified potyvimsUnidentified potyvimsUnidentified potyvimsUnidentified isometric vimsPeanut mottle potyvirus

of other potyviruses unrelated to BCMV or anyother known potyvirus were obtained from P.vulgaris and various wild legume species duringthe survey. An isolate from Cassia occidentalis(synonym Senna occidentalis) obtained in Ethio-pia may be a previously undescribed potyvirus,and has been tentatively named cassia severemosaic virus (Walkey et al., 1994) (Table 10).

Host range

A total of 29 legume species were tested for theirsusceptibility to standard strains of BCMV. Fivespecies, namely Cassia didymobotrya, Crotalarialaburnifolia, Desmodium heterocarpon, D. tri-florum and Rhynchosia sublobata, were appar-ently resistant to infection by all strains tested. Afurther five species, namely Centrosema pubes-cens, Crotalaria anagyroides. C. lanceolata, C.ochroleuca and Rhynchosia minima, were suscep-tible to infection by five of the BCMV strainstested (Table 11). Twenty-two species wereapparently resistant to NLl, but many specieswere susceptible to the other four BCMV strains,particularly NL3. Only Crotalaria anagyroides,C. incana and Vigna angularis were susceptible toIsolate 30, an isolate of a novel pathotype derived

from C. incana growing in Uganda. In manycases infection was symptomless and onlydetected by ELISA.

Aphid transmission

The aphid transmission of isolates 28, 30 and 38of a novel pathotype from Crotalaria incana(propagated in Chenopodium quinoa), isolate 145(pathotype IVb) from a Rhynchosia species(propagated in P vulgaris) and isolate 499(pathotype Via) from Macroptilium atropurpur-eum (propagated in P. vulgaris) was investigated,using short acquisition and inoculation accessfeeding times, to Nicotiana ctevelandii, Chenopo-dium quinoa, P. vutgaris cvs The Prince andDouble White, Crotataria incana. Cassia occi-dentatis and C. sophera (Table 12).

Isolates 28, 30 and 38 were aphid-transmittedin a non-persistent manner to Crotalaria mcana.Cassia occidentalis, bean cv. Double White andC. quinoa. Isolates 28 and 30 were non-persistently aphid-transmitted to Cassiasophera. Isolates 145 and 499 were only testedfor aphid transmission to cv. Double White, andthey were both non-persistently aphid-transmitted to this cultivar (Table 12).


Table 11. Reactions of wild legume species to inoculation with bean common mosaic virus strains and isolate 30

Host species

Cassia didymobotryaCassia hirsutaCassia occidentalisCassia sopheraCajanus cajanCentrosema ptumieriCentrosema pubescensCrotataria anagyroidesCrolataria goreensisCrotataria incanaCrotataria junceaCrotataria taburnifotiaCrotalarta tanceotataCrotataria ochroteucaCrotataria retusaCrotataria verrucosaDesmodium heterocarponDesmodium triflorumGtycine maxGtycine tomentettaIndigofera hirsutaMacroptitium atropurpureumRhynchosia diversifotiaRhynchosia edutisRhynchosia mutimaRhynchosia subtobataVigna angularisVigna radiataVigna unguicutata

Seed source

EthiopiaUgandaEthiopiaEthiopiaYemenColombiaColombiaColombiaColombiaColombiaColombiaColombiaColombiaColombiaColombiaColombiaColombiaColombiaUSAColombiaUgandaColombiaColombiaColombiaColombiaColombiaYemenYemenYemen

7SJL1

0mos

0000

mosvc/E

0000

0/Emos

0000000000

mos0

mot00

NL3

0mos/Emos/E

0/E0/E0

mosvc/E

0vc/E0/E

0vc/E

mos/E0

0/E00

cl/E0/E0/E0

0/E0/En/E

00/Emot0/E

NL4

ntEntnt0

0/Emosvc/E0/E

00nt

0/E0/E0/E

000

0/E0

0/E000

n/E0

0/E00

NL6

0mos/B

00/E0/E0

mosVC

0cl00

0/E0/E0

0/E0000

0/E000

n/E0

mot /Emot/E

0/E

NL8

00

mos/E0/E0

0/Emosvc/E

0vc/E

00

0/E0/E

0000

0/E0 E0/Emos0/E

0n/E

000

0/E

30

0000000

VC

0vc/E

0000000000000000

mot/E00

0, no symptoms; mos, systemic mosaic; vc, systemic vein clearing; cl, systemic chlorosis; mot, systemic mottle; n,systemic necrosis; /E, virus detected by ELISA using the monoclonal antibody 197; nt, not tested.

Seed transmission

Of the 29 legume species inoculated with thestandard BCMV strains (Table 11), only sevenproduced seed. Some plants failed to flower, andothers flowered but either fruits were notproduced or they contained no seed. Of theplants which did produce seed, only a lowpercentage germinated in some cases to producerelatively few seedlings. If many seedlings wereproduced, fifteen were tested by ELISA for seed-transmitted virus; for species where fewerseedlings were produced all of them were testedfor the presence of virus. Plants of Crotatariaochroteuca. Cassia Occidentaiis and C sopherathat produced seed were those inoculated withNL3 and NL8; in each case a proportion of theresulting seedlings was infected (Table 13). Allinfected plants of Crotalarta incana producedseed, and a proportion of the resulting seedlings

from plants infected with NLl, NL3, NL4, NL6,NL8 and isolate 30 was infected (Table 13).

All the BCMV strains tested were seed-transmitted to a proportion of seedlings ofMacroptilium atropurpureum, although duringhost-range studies only NL8 was found to infectM. atropurpureum as demonstrated by testingwith ELISA (Table 11). This could be explainedby the concentration of virus in the parent plantsbeing too low to be detected by ELISA.

In some cases, symptoms in bean cvs. DoubleWhite and Sutter Pink were so severe that noseed was produced. Alternatively, seed wasproduced by one cultivar but not the other(Table 14). There was no seed production byeither cultivar infected with isolates 28, 30, 38,499 or 963. In contrast, both cultivars producedseed when infected with isolates 820 and 830 Inthe cuse of the latter isolates, seed from cv. SutterPink was not infected but a proportion of the

540 N. J. Spence &D.G. A. Walkey

Table 12. Aphid transmission of virus isolates from wild legume species to various hostspecies

Host species

Cassia occidentalisCassia sopheraChenopodium quinoaCrotalaria incanaNicotiana ctevelandiiP. vulgaris cv. The PrinceP. vutgaris cv. Double White

28

2/2 ml/2v2/2c2/2v0/20/22/2n

30

2/2ml/2v2/2c2/2v0/20/22/2n

Isolate

38

l/2m0/22/2c2/2v0/20/22/2n

145

ntntntntntntl/4m

499

ntntntntntnt2/4

Nominator = number of seedlings developing symptoms; denominator = total number ofseedlings tested.m, mosaic; v, vein clearing; c, chlorosis; n, necrosis; nt, not tested.

seed from cv. Double White was infected. Viruswas not transmitted in seed from plants of cv.Double White infected with isolates 465 or 956.Isolate 145 was seed-transmitted in cv. SutterPink, but no seed was produced by plants of cv.Double White infected with this isolate.

DISCUSSION

Geographical distribution of BCMV

BCMV isolates were assigned to pathotypesaccording to their pathogenicity phenotype ondifferential host cultivars, and could readily beclassified as belonging to either the 'A' or 'B'serotype by ELISA. Pathotype and serotypewere strongly correlated. The results clearlydemonstrated the widespread occurrence andpredominance of the 'A' serotype in central,eastern and southern Africa; this serotype wascompletely correlated with the occurrence of atemperature-independent necrotic response bycultivars carrying the / gene.

The widespread occurrence of isolates similarto NL3 and NL8, and of other isolates of the 'A'serotype with novel pathogenicity phenotypes,emphasizes the importance of incorporatingresistance to these so-called necrotic strains intonew cultivars. Many cultivars that have beenintroduced into bean improvement programmesin Africa carry only the dominant / resistancegene and are therefore susceptible to 'black root'when infected by any isolate that provokesnecrosis in the presence of this gene. In areaswhere necrosis-inducing isolates exist, it isessential to incorporate additional resistance

genes such as /)c2^ or bc3 into breedingprogrammes. Exceptionally, necrosis-inducingisolates of the 'A' serotype were not located inEthiopia. However, three isolates of a novelpathotype were found that did induce necrosis inhost group 8 cultivars even though they were ofthe 'B' serotype. In Ethiopia, cultivars carryingthe / gene alone may be sufficient to conferresistance against BCMV in most areas, butplant breeders need to be aware of the existenceof this novel pathotype, which could influencethe durability of any newly introduced resistantbean cultivars.

The occurrence of non-necrosis-inducing iso-lates of the 'B' serotyp)e in both beans and wildlegume species, over a relatively wide geographi-cal area around Kisii and Kakamega in westernKenya, is of interest. One isolate expressed aphenotypic reaction pattern identical to patho-type Va, while others expressed novel pheno-types. NY 15 is the type strain of pathot>pe Vawhich until now had only been recorded in NorthAmerica.

The widespread occurrence and predominanceof pathotypes which induce necrosis in cultivarscarrying the / gene and which are of the 'A'serotype raises questions about the geographicalorigin of these pathotypes. NL3 and NL5 wereisolated from cultivars carrying the / gene in beanvariety trials conducted by a commercial com-pany which at that time was not multiplying beanseed in Africa (Hubbeling, 1963.1972). However,as cultivars carrying the / gene do not transmitvirus in their seed (Morales, 1989), they wereprobably not the source of the virus. It is morelikely that the virus was aphid-transmitted from


Table 13. Seed transmission of standard bean common mosaic virus strains and isolate 30 in wild legume species

Host species

Cassia occidentatisCassia sopheraCrotataria incanaCrotalaria lanceotataCrotataria ochroteucaMacroptitium atropurpureumRhynchosia diversifotia

Seed source

EthiopiaEthiopiaColombiaColombiaColombiaColombiaColombia

NLl

ntnt4/150/14nt3/155/5

NL3

2/53/52/150/152/54/152/2

NL4

ntnt1/15ntnt9 152/2

NL6

ntnt1/150/6nt1/7nt

NL8

2/52/53/150/62/5

10-154/5

30

ntnt5/15ntntntnt

Nominator = number of seedlings developing symptoms; denominator ••Isolate 30 was obtained from Crotataria incana (Uganda).nt, not tested as no seed produced.

total number of seedlings tested.

different cultivars included in the trial from otherseed companies. These other companies wereknown at that time to be multiplying seed inAfrica likely to have been included in these trials(L. Bos, personal communication, 1993). NL8was isolated from the progeny of a breedingexperiment in a private breeder's field in Holland(Drijfhout & Bos, 1977). This strain may alsohave been introduced from Africa via the sameroute as NL3 and NL5, but there is no furtherevidence to confirm or discount this possibility.

The geographical origin of isolates hasparticular significance in view of the recentproposal that the so-called necrotic strains aresufficiently distinct from other forms of BCMVto be regarded as a separate virus designatedbean necrosis mosaic virus (BNMV) (McKern etat., 1992a, 1992b; Vetten et at., 1992). Necroticstrains are not thought to occur naturally inSouth America, the centre of origin of P.vutgaris, and when they have been recordedthere, or in North America, their occurrence canusually be traced to infected imported seed (F.Morales, personal communication, 1990). Theresults of this study support the contention thatthe two serotypes are distinct and may haveseparate origins. The prevalence of isolates of the'A' serotype in central and eastern Africaindicates that they may have evolved in thisregion, whereas the 'B' serotype of BCMVprobably has its origin, along with P. vutgarisbeans, in Central or South America.

The occurrence of the 'A' serotype in wildlegume species in Africa, and the observedtolerance (symptomless infection) of some ofthese species, is a further indication that theirgeographical origin may be central and southernAfrica. The phenotypic and therefore genotypicdiversity of pathotypes from this region suggests

that the host population imposes a strongselective force for pathotypic variation. Poty-viruses are known to produce new variants byrecombination among heterogeneous RNApopulations within infected plants (Goldbach,1992; Lecoq & PurcifuU, 1992). In this context,the recovery of isolates expressing a previouslyunrecorded pathogenicity phenotype is of inter-est. The majority of the isolates expressing novelphenotypes induced a temperature-independentnecrotic reaction in cultivars carrying the / gene,and were of the 'A" serotype. The pathogenicityphenotypes of some of these isolates aresummarized in Table 6, and compared withthose of NL3 and NL8.

A range of wild legume species that commonlyoccur in Africa, notably of the genera Cassia,Crotaiaria. Macroptilium. Rhynchosia and Vigna,were susceptible to infection by a number ofstandard strains of BCMV. Some species wereparticularly susceptible to NL3, which wasprevalent in the areas where these genera occur.Infection was often symptomless and onlydetected by ELISA. Such latent infectionsimply genetic adaptation by the host as a resultof host and pathogen coexistence over a longperiod of time. This suggests that some patho-types of BCMV may have originated in thisregion of the world.

Several BCMV isolates from wild legumespecies were aphid- and seed-transmitted toother legume species and P. vulgaris. andthe standard BCMV strains were also seed-transmitted in wild legume species. It is there-fore possible that in bean-growing areas inAfrica, BCMV could be transmitted fromcultivated or wild legume species to beans, orfrom beans to the other legume species. Infectedwild legume species could provide a source of


Table 14, Seed transmission of bean common mosaic vims isolates fromwild legume species in P. vulgaris cvs Double White and Sutter Pink

Isolate

145465820830956

Double White

Mosaic'

nt'0/73/111/100/13

ELISA"

nt0/73/111/100/13

Sutter Pink

Mosaic

4/8nt0/150/10nt

ELISA

4/8nt0/15O/IOnt

Nominator = number of infected seedlings; denominator = number ofseedlings tested.' Seedlings with mosaic symptoms."" Seedlings which were positive for BCMV in ELISA.' nt, not tested as no seed was produced.

inoculum to infect bean crops, which are growntwice a year in some areas. They might also act asalternative hosts, facilitating the evolution ofpathogenic variants of BCMV.

A number of isolates expressing novel patho-genicity phenotypes were also found to inducetemperature-dependent necrosis in the differen-tial host cultivars carrying the / gene; thepathogenicity phenotyi>es of these isolates arecompared with those of NL6 in Table 7. Fewerisolates with novel pathogenicity phenotypeswere of the 'B' serotype and did not inducenecrosis, and all but one of these isolates werefound in western Kenya. The reactions ofdifferential hosts to these isolates are comparedwith those of NY 15 in Table 8.

The gene-for-gene relationship described byDrijfhout (1978) to explain the genetic relation-ship between BCMV and differential hostcultivars of P. vulgaris (Table 15) goes some ofthe way towards providing a genetic interpreta-tion of the novel pathogenicity phenotypesobserved in this study. It does not, however,provide an explanation for the full range ofpathogenic variation observed among isolates ofthe 'A' serotype. Drijfhout (1978) presented thetotal number (64) of virus genotypes that weretheoretically possible based on combinations ofpathogenicity genes P\, P\^, P2 and P2- (Table15). It was assumed that all pathogenicity genesoccurred at different loci in the virus genome andcould therefore, be expressed by a single virus. Itshould be noted that no isolate carrying theputative pathogenicity gene P^ to match hostgene bd has been found to date. At the time ofDrijfhout (1978), only seven of the 64 possible

theoretical genotypes had been identified, andonly three strains (NL3, NL5 and NL8) expres-sing temp>erature-independent necrosis on / genecultivars were known.

The strains Drijfhout assigned to pathotype FVwere considered to carry identical pathogetiicitygenes (Drijfhout, 1978), but the pathotype wassubdivided into those strains that do not inducenecrosis in / gene cultivars (IVa) and strains thatdo (IVb). Pathotypes V and VI were similarlydivided. The gene-for-gene model described byDrijfhout does not provide an explanation forthese differential reactions on / gene cultivars. Itis proposed that there may be a virus generesponsible, in combination with the / gene, forinducing temperature-independent necrosis (N),designated Px, and a gene responsible forinducing temperature-dependent necrosis (n),designated Px'.

The theoretical Px and Px^ pathogenicitygenes are included in the proposed genotypes ofthe temperature-independent and temperature-dependent necrosis-inducing BCMV strains;NL8, NL6/US5, NL2/US2 and NL3/NL5(Table 16). If Px and P.r are not allelic, the Mtheoretical combinations o( P\, P\', P2 and P^are expanded to result in 256 possible virusgenotypes. If this hypothesis is correct, theBCMV isolates expressing novel pathogenicityphenot>p>es could be carrying the combinationsof PI. P\' PI, P2-, Px or Px' pathogenicitygenes listed in Table 16. It should be noted thatsome isolates induce either a temperature-independent necrotic reaction or a temperature-dependent necrotic reaction in all host cultivarsof groups 8 and 9. This indicates that the isolates


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Table 17. Theoretical pathotypes of novel isolates of bean common mosaic virusisolated from Phaseolus vulgaris and other legume hosts in different countries in Africa

Country

BurundiEthiopiaKenyaLesothoMalawiRwandaSwazilandTanzaniaUgandaZaireZambiaZimbabwe

Total

Number of isolates assigned to each theoretical

VI'

100120020001

7

V2

0I0020010010

5

V3

020000000000

2

V4

100001003100

6

V7

202003130101

13

V9

000001000000

1

pathotype of BCMV

V12

106021000100

11

Total

53g166163312

45

" See Tables 15 and 16 for theoretical virus pathotypes.

carry either Px or P.x^ In contrast, other novelisolates appear to induce a temperature-indepen-dent necrotic reaction in host group 8 cultivarsand a temperature-dependent necrotic reactionin host group 9 cultivars (see isolates 127 and 179in Table 7). It is proposed that these isolatescarry both Px and Px^ The pathogenicitygenotypes suggested by the phenotype of novelisolates found in this study can be matched tosome of the theoretical genotypes described inTables 15 and 16 (Table 17).

The results of this survey do not provide proofof the existence of pathogenicity genes Px andPx^ Further genetic studies are required todetennine the genetic basis of differentialnecrotic host reactions. The response of cv.Jubila (group 9a) was frequently inconsistentwhen it was inoculated with isolates that induce atemperature-independent (N) necrosis in allother group 8 and 9 cultivars (Tables 5-7).This cultivar often responded by developingnecrotic symptoms following inoculation withthese isolates only at 32'C and not at 26°C. It ispossible, therefore, that there may be anadditional gene or genes in this cultivar thatmodifies the development of the necrotic symp-toms, or that local fluctuations in temperaturemay influence gene expression.

All the isolates characterized during this studyhave been stored in liquid nitrogen and in freeze-dried material at HRl, Wellesbourne; they areavailable to other workers on request. Many

isolates have already been returned to theircountry of origin for use during selection forresistance in national breeding programmes.Because of the considerable variation that existsbetween local isolates and standard BCMVstrains, the use of local isolates is consideredmore appropriate for screening local germplasm.

ACKNOWLEDGEMENTS

We thank personnel from the CIAT RegionalBean Programmes and National Bean Pro-grammes in Africa for their assistance in thesurvey work. We also thank Dr G. I. Mink andDr H. J. Vetten for antibodies, Mr R. Sampsonfor photographs, Mrs J. M. Bambridge fortechnical assistance, Dr D. A. C. Pink forhelpful discussions, and Dr I. R. Crute forcritical review of the manuscript. The GeneticResources Unit at CIAT, Colombia, kindlyprovided legume germplasm. The work wasfunded by the Overseas Development Adminis-tration through the Natural Resources Institute.BCMV isolates were imported into the UKunder MAFF Licence No. PHF 1227A/39(60).

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