PROCEEDINGS OF THE INTERNATIONAL SYMPOSIUM

ON MYCOTOXINS

SPONSORED B Y F O O D A N D D R U Q A D M I N I S T R A T I O N

ROOKVILLE. M A R Y L A N D AND

N A T I O N A L RESEAROH O E N T R E OAIRO, E G Y P T

1983

Aflatoxin Production in Groundnut Cultivars Resistant and Susceptible t o Seed Invasion by

Aspergillus flavus"

V.K. RlEHAN and D. McDONALD

I n r e n t a t i a t ~ l C r o p s Rcscarclr Ins / i t t t re for rhe Serni -Ar id T rop ics (ICRISAT), Groundnur I t t r p r o ~ ~ e r n c ~ t t P rogra tn , Pafartchcru P - 0 . 5 0 2 324 , A .P., I r td io

A h a l r n c t 7 % ~ IISC qr grorrndnrtt r~1tivar:r re.ri.rtartt ro seed irrvasiorr nnd colonrzarron by A.rpcrgillr~~ /?n,.r!s is a po.rsible nrrntts o f p r r ~ m t i r r ~ or reducing apatoxirr conrorrrinariorr. Such rrsi.rrnncr war identified in .ret~eral rulri~wrs. one ojw.hrrh was r/tc releared contrrrcrriol lrrllinn rrrlrivnr JII . N o direr1 rclorronship rvor filrrnd brrn.cm rrsirtnrrrc ro .reed mlorriznion by A. flovrnr orrd Ihc qrmr rrrj, of a/lotorhts p rod~crd niren sevdr wrrr colorrized by m.rigrnic strains o/ rhc frmgrrs. Sorr of rhc rrrlril~ars with scrd rc.ri.rmnce lo A. flnvrts colonimlion have been formd reoisrnnt ro f i r n ~ a l pod rot.

I n t r o d u c t i o n

Contamination o f groundnuts with aflatoxin is n serious problem in n ~ a n y parts of the world. The use o f groundnut cultivars resistant t o seed invasion and co lon iza t io~~ hy toxigenic strains of Aspergi l l t ts flavrrs has been suggested as an effective mcans of preventing o r at least reducing aflntoxin conta~ninntion (1-3). Early research reported varictal differences in aflatoxin production when autoclaved seeds were colollized by toxigenic strains of A . J7a1.rts (4,s). Alth- ough claims of resistance t o aflatoxin production were not confirmed by later research (6,7), quantitative varictal differences in aflatoxin production were indicated.

This paper reports on screening of germplasni for rcsistancc lo seed invasion and colonization by A. f l a v u ~ and for resistance to aflatoxin production when seeds are colonized by toxigenic strains o f the fungus.

Submilted a% Conrcrcncc Pewr No. CP-49 by thc Inlcrnalionnl Cropq Research lns- titute Tor the Semi-Arid Tropics (ICRISAT), Patancheru P 0 502 324, A .P , India

222 V.K, MElIAN AND D. MCDONALD

M a t e r i a l s a n d M e t h o d s

Seeds of all cultivars were obtained from the 1979 and 1980 rainy season and the 1979 -80 and 1980-81 post-rainy season crops grown a t ICRlSAT Center, Patancheru, A.P. , India, The crops were not subjected to drought strcss. Cultivars were harvested at opti~num maturity nncl plants werc arranged in windrows ill the field with pods exposed. Aftcr windrow-drying for 2 days in the post-rainy and 3 days in the rainy seasons, thc pods were hand-picked and sun-dried on mats until seed moisture contents were reduced to !ess than 7%. Pods were then stored in cloth bags in the laboratory until seeds were required for testing.

Seed Colortizoriort Test

Tllc Mixoti arid Rogcrs nic[liod ( I ) wit11 sonic ~iiodilication (8) was uscd. For each cultivar, 3 plates of surfacc-sterilized (sonkcd in 0.1% aqueous solt~tion of niercuric chloride for 2 min f'ollowcd by 4 rinscs in stcrile distilled water) hydrated sceds were inoculated with a spore suspension (4 - 0 x 10" condia Iml) of the toxigenic A. flnt7lrs strain AF8-3-2A. The plates wcre incubated at 25°C aad percentages of seeds colonized were recorded aftcr 8 days.

I

9\

Aflotoxi11 PI ot1~rct.011 Test

Using seed from the 1980 rainy season crop, aflatoxin production tests nrcrc carried out on each cultivar by the mctl~od described hy Mehan and McDonald (8). Surfnce-sterilized secds in 3 plates had their testas damaged by e*lapinp ti1e11i with a stcrile needle. Tlie secds were then inoculatcd nrith a spore suspensior~ of A . . f lnr~w strain AFS-3-2A and incubated as described above for the seed colonization test. After 10 days of incubation, atlatoxins were extracted using Romer's method (9), determined quantitatively by the method of Nabney and Nesbitt (lo), and expressed as micrograms per gram of' seed.

R e s u l t s a n d D i s c u s s i o n

Frorii 150 cultitlars scrcencd, 8 showed resistance to seed colonization. Mean sceci coloni7ation pcrccntngcs nrc shown in Tablc I for tlic S resistant cultivars, and 2 control cultivars, the susceptible T M V 2 and the highly suscep- tible 0G43-4-1, for seeds from 2 rainy season and 2 post-rainy season crops. The cultivnrs Ah 7223, Monir 240-30, and U F 71513 showed the higlicst resis- tance. Less resistant, but still significantly niore resistant than TMV 2, were cultivars J 11, Faizpur, Var. 27, PI 337394 F, and PI 337409. PI 337394 F and PI 337409 have been reported resistant in the United States and in Senegal (2). These 2 cultivars are not agrononrically acceptable but J I I is a released commcrci:~l culti\-ar in India. This cttltivnr nnd t l ~ e othcr A. /7nvrrsresis- tant cultivars Ah 7223, hlonir 210-30. and Var. 27 have also been fbund resis- tant to a fungal pod rot a t ICRlSAT Center.

Proc. Int . Sytrtp. Mycotoxins (1982)

Aflatoxin Production in Groundnut Cultivars Resistant and Susceptible t o Seed Invasion by

Aspergillus javusw

V.K. RlEHAN and D. IklcDONALD

Inrcrtiatia~rl Crops Resrarcli Jn.rtitttte for the Semi-Arid Tropics (ICRISAT), Groundnut hrpro~~entmt Prograrn, Pa!orichcru P .0.502 324 , A .P., bidio

Tlrr rrsr of grortrrdnrrt crrlrivo~r resistant to seed irrrasior: and coloniza~ion by AspergNlrrr f7a~us is a pssihle nrmrts of pre~'~nrirrg or reducing a~otosirr conroirrinoriorr. S~cch rcsi.rmncr wur ihntificd in se~~eral crilfit*ar~, onr of w.hic/r a~os tltc rcienscd corrrtr~crciol lrrt/ion crrltivnr J l l . No direct relationship ~i*as furtrtd bcr~i~rrn rrsisfotrcr to sred roloniznion by A.Jlnvns ortd /he qrmr tity of pflato.rirr.r prodict.rd n,/rar serdr nscrc colonized I?)* to.u~rrric strains of fhe Jtcngrrs. Sorr o j thr crrltivars ~virh srrd rc~istancc fo A. /lavrrs colonizn/ion hove been fortnd reaistnnt to finpal pod rot.

I n t r o d u c t i o n

Contamination of groundnuts with aflatoxin is a serious problem in many parts of the world. The use of prouudnut cultivars resistant to seed invasion and colonizatiot~ by toxigenic strains of Aspergillus flovus has been suggested as an effective means of preventing ?r at least reducing aflatoxin contalilinntion (1-3). Early research rcported varictal dilTerences in aflatoxin production when autoclaved seeds were colonized by toxigenic strains of A. jl'a~~t~s (4,5). Alth- ough clairns of resistance to aflatoxin production were not confirmed by later research (6,7), quantitative varietal differences in aflatoxin production were indicated.

This paper reports on screening of germplasnl for resistance to seed invasion and colonization by A. Javu.r and for resistance to aflatoxin production when seeds are colonized by toxigenic strains of the fungus.

- ---- Submitled as Confcrcncc Paper No. 0 - 4 9 by thc Intcmnlionnl Crops Rcscnrch Ins- tilule for the Semi-Arid Tropics (ICRJSAT), Patancheru P.0.502 324, A .P.. India

222 V.K, MEtIAN AND D. MCDONALD

M a t e r i a l s a n d M e t h o d s

Seeds of all cultivars were obtained from the 1979 and 1980 rainy season and the 1979 -80 and 1980-81 post-rainy season crops grown a t ICRlSAT Center, Patancheru, A.P., India. The crops were not subjected to drought stress. Cultivars were t~nrvested at optitiium ninturity :~nd plants were arranged in witidrows il l tlie field with pods exposed. Aftcr windrow-drying for 2 days in the post-rainy and 3 days in tlie rainy seasons, the pods were hand-picked and sun-dried on mats until seed moisture contents were reduced to less than 7%. Pods were then stored in cloth bags in the laboratory until seeds \\.ere required for testing.

Seed Colonization Test

Tlic Mix011 arid Rogcrs niclliod ( I ) nitll sot~ic riiodificntioti (8) wns used. For each cultivar, 3 plates of surface-sterilized (soaked in 0.1% aqueous solution of niercuric chloride for 2 min iollowcd by 4 rinscs in sterile distilled water) hydrated sceds were inoculated with a spore suspcnqion (4 -0 x IF condia /ml) of the toxigenic A. flai+trs strain AF8-3-2A. Tlie platcs were incubated at 2 5 O C and percentages of seeds colonized were recorded after 8 days.

I

.I\

Affotoxirr P~odtrct.nrt Test

Using seed from the 1980 rainy season crop, aflatoxin production tests were carried out on each cultivar by the mcrllod described by hlehan and h4cDonald (8). Surface-sterilized seeds in 3 plates had their testas damaged hy rTl.aplng them with a sterile needle. Tlie seeds were then inoculated with a spore suspetisinti of A . j/orlrs strain AF8-3-2A and incubated as described above for the seed colonization tcst. After 10 days of incubation, atlatoxins were extracted using Romer's method (9), determined quantitatively by the method of Nabney atid Nesbitt (lo), and expressed as micrograms per gram of seed.

R e s u l t s a u d D i s c u s s i o n

Frorn 150 cultivnrs screened, 8 showed resistance to seed colonization. hlenn sccd colonizntion pcrcclitngcs nrc slio\vn in Table I for tlie 8 rcsistotit cultivars, and 2 control cultivars, the susceptible TMV 2 and the highly suscep- tible 0043-4-1, for seeds from 2 rainy season and 2 post-rainy season crops. The cultivnrs All 7223, Monir 240-30, and U F 71513 slio\ved the higlicst resis- tance. Less resistant, but still significantly niore resistant than TMV 2, were cultivars .l 1 I , Faizpur, Var. 27, PI 337394 F, and PI 337409. PI 337394 F and PI 337409 have been reported resistant in the United States and in Senegal (2). These 2 cultivars are not agrononiically acceptable but J I I is a released comtne~.ci:il culti\*nr in Intlin. This citltivar nnd tlie other A . ,f?ovrrsresis- tnnt cultivnrs Ah 7223, hlonir 2.10-30. and Var. 27 have also been found resis- tant t o a fungal pod rot a t ICRlSAT Center.

Proc. In t . Sgmp. Alg~cotoxins (1982)

GROUNDNUT CUIATIVAItS 223

I t is evide~it that levels o f secd co lo~i izn l ion o n n l l cultivnrs were liiglicr on sccd iron1 post-rainy ficnso~i crops 1lia11 011 scctl rroln r n i ~ i y scnson crops. Fluctuation in soil moistt~re during pod de \~c lop~nc~ i t nnd i i i g l ~ tcnipclnturcs durittg post-harvest dryiqg o f t l ~ e irrigated post-rniny seeson crop linve been suggested as possible reitsons for l l i is diRerctice ns tl~cse factors could cnllsc damage l o l l ~ e rrccd tcatas. This is bcing invcslig:~tctl.

Early I-cporls o f rcsistr~~ice to i~ f ln lox i l i ~ ~ r o r l t ~ c t i o n (4,5,12) wcrc not subs- tantiated but indicntions wclc obtninccl of pos5il)lc gctictic dimcrcrices ill nrlioulits o f allatoxil l p r 0 d u ~ ~ d (6,7,13), l 'oblc 2 gives aflntoxill 11, production fipurcs for 7 o r tlrc A, flnvra-rcsisti~iit cultiv:lrs, for l l ic susccptihlc I 'M\' 2, ntld the highly susceptivle OG-43-41 n ~ i d FESIt-11-PII-U2-Il l. A l l cultivars

TABLE 1. Percent sccd culonirn(ion Ipy A . /lol,rts o l sclcclcd rcsialnnt nnd s~~sccpflble cultivnr~

Proc, I r t t . Syinp. Mj*cotoxinr (1982)

CnIII\nr

WF 71513 . . . . PI 337394 F . . PI 337409 . . J 1 I . . . . .

4 )

Ah 7223 . . . .

hionir 240-30 . . Var.27 . . . . . . Faizpur . . . . TMrV 2d . . , . 00 43-4-1' . . .

--. -. -- CD (a1 5%) . .

Suurcc

USA

Argentine

Argenliria

lnclia

Nipe~ia

Australia

India

India

lndin -- -----

'

cv (%) . . . I I i9 1 1 9 i I ' !

! I - I ---- I 8 R

I I'erccnl sccd ~olonlzctl

- - - - - - - - - . - - - . . - Rnitrg sonsults Po*!-rn!ny scnsotls

0 Mcan of 3 rrplicotions. - . - 1101 lcqtcrl

1) Susctplible chcck CV, c llighly suwcl)lihie CV

-- .- . - - I . - - - 1 9 8 U _

-

14 2

19 5

21 0

1 7 . 5

I 5 8 8

I4 3

19 9

19 1

41 5

98 2

4 2

1979 __-

--

7 2

8 8

9.8

- - -

-

34 8

- ' 1980 1979180 -. -__

6 6

R I

9 4

1 2 n

4 5

9.0

3 4 9 1 9 1 2

. -- --. - .

- ----

13 1

19 9

20.8

21.8

I5 7

I 6 I

96 9 . *. - +- . -

1 4 1 ---4- (, 3 2

9 7 ' 212

9 H

36 7

19 7

49 4

G R O U N D N U T CULTIVARS

TMV 2 ] -0- P I 337409

--t- J 1 1

1

08)s of incubdtion f o l l o w ~ n g inoculat lor with toxigenic A , flaws strarn Are-3-2h

Fig. 1, Aflatoxrn B, accumulalion in inoculated seeds or 4 cultrvar~ inciibated at 25 3°C.

R e f e r e n c e s

(1) Mlxon, A.C., & Rogers. K.hl. (1973) Okn~irrerrx 28, 85-86.

(2) Zmbettrkls, Ch., BockcllcMorvan, A., Wal ip , F. & Rossion, J. 11977) OI+aipitrccr.y 36, 377-385.

(3) Mthnn, V.K., McDonnld, D., Niplam, S.N., & l.alitha, R. (1981 ) Ol&a~irrctrx 36. 501-507.

(4) Kulkarnl, L.C., Sharief, Y., & Sarmn, V.S. (IM7) It~rlmtf rnrnrrtrji 17, 1 1-I?

(5) RRO, K.S., & TupuIc, P.G. (1967) h'nturr 214, 738-739.

Proc. Int. Synrp. Alycofoxinr (1982)

226 V.K. M E I . I A N A N D D.h.(cDONALI)

(7) .\uila. S.S., C'l~ohnn, J.S., & hlehan, V.K. (1978) prrrijoh . Ier ic, I11ii13. J. Rrs. 15. 400-~l03.

( Y ) h l c h n ~ ~ , V.K., & hlrl):~nnld, 1). (19.90) IC KIS 4 T <;rofr!r Ifrrrr Irrrlrrov:'~~r~"'r A.o~.er,v.rr Orcvairrrrrrl Ptrprr. 2. I - I5

(9) Homcr, 'T.R. (1975) J Assor. Off . Arinl. Chcrrr. 58. 500-506.

( 10) Nnbncy, .I., L Rcshitl, B.P. (1965) Arlrrl~..ir 90. 155.160

( I I ) hlclr: i~~, V.K., h~cI)onnld, D.. & Hno, V.II. (1981) Prar. Irfr. Pcottrrr R1.s. E:lrrr. Sor. 13 ( 1 ) . 91.

(12) l'11pu1c. P.G.. nbftl, R.V., & Xnpnroinn. \'.T. f197i) Al.r.h. I~rsr. Pnsrerrr ?rmis 54. 487-491,

(13) hlchtln, V.K., hlcDonald, D., R. (;lbbatla, H.lY f 1981) 0lroginr~rr.y rn p r t . . ~

Epidemiology and control of groundnut bud necrosis and other diseases of legume crops in India caused by tomato spotted wilt virus

D . V . R . R E D D Y , P .W. AMIN, D . McDONALD & A . M . G H A N E K A R Groundnut Improvement Program. International Crops Research Institute for the Semi-Arid Tropics IICRISATI, Patancheru P.O.. A.P. 502 324. India

INTRODUCTION

Tomato spotted wilt virus (TSW) was first reported in India in tomato in 1964 (Todd et al., 1975). The occurrence of T S W on a legume in India was first recorded in 1968 (Reddy et al., 1968). The "bud necrosis disease" of groundnut, caused by TSW, is now considered to be one of the most damaging groundnut diseases in India (Ghanekar et al., 1979a; Reddy, 1980). Bud necrosis is likely to have been present in India for some time although it has only recently become economically important. T S W has also been reported on groundnuts in Brazil (Costa, 1941), the United States of America (Halliwell & Philley, 19741, South Africa (Klesser, 1966) and Australia (Helms et dl., 1961). This chapter considers the epidemiology and control of bud necrosis and gives a brief account of other economically important diseases of legumes in India caused by TSW.

OCCURRENCE AND DISTRIBUTION OF LEGUME DISEASES CAUSED BY T S W IN INDIA

Our surveys show that bud necrosis is widely distributed in the main groundnut-growing regions of India and that it is endemic in the states of Andhra Pradesh and Tamilnadu. Extensive infection has also been seen in parts of the states of Maharashtra, Gujarat, Rajasthan and western Uttar Pradesh. The greater incidence of bud necrosis in recent years may be related to the expansion of irrigation projects which has led to continuous cropping of groundnuts and other hosts of TSWV. Until recently most groundnuts were grown in the rainy season but increased demand has caused an expansion of the post-rainy season, irrigated crop.

The economically important leaf c'url diseases of green and black gram (Vigna radiata) (Nene, 1972) have recently been shown to be caused by TSWV (Ghanekar et dl., 1979b) and field trials showed that TSWV can also cause economically important diseases of pea (cultivar Bonneville), broad bean (cultivar Local), cowpea (cultivar C-152) and soyabean (cultivar Bragg) .

Plumb R.T. &Thresh J.M. (1983)Plant Virw Eprdrm~ology. Blackwell Scientific Publications. Oxford.