Mycotoxin Research. Vol. " ('995)

Preharvest aflatoxin contamination of groundnuts subjected toterminal drought stress In postrainy season'

V K Mehan·, N Ramakrishna, R C Nageswara Rao, and D McDonaldLegumes Program, International Crops Research Institute for theSemi-Arid Tropics, Patancheru PO, AP 502 324, India* Corresponding author

'ICRISAT Journal Article No. JA-1446.

Abstract

Groundnuts grown in the postrainy season under terminal droughtstress imposed by withholding irrigation, or under a water-deficitgradient created by line-source sprinkler irrigation, were examinedfor preharvest aflatoxin contamination. High levels of aflatoxin 8,were found in damaged seeds in both situations. When grown undercontinuous drought-stress, toxin levels in damaged seed samplesranged from 1480 to 2467 ~glkg in the 1990/91, and 1.3 to 2000 ~glkg

in the 1991/92 postrainy seasons. Aflatoxin 8, contamination in alldamaged seed samples increased with increasing water deficit; toxinlevels ranged from 26 to 850 ~glkg across the water deficit gradient.Aflatoxin was either absent or almost negligible (1-2 ~g/kg) inapparently undamaged seed samples. Low risk of aflatoxincontamination in apparently undamaged seeds of groundnuts grownin postrainy seasons is indicated, even when there is terminaldrought stress.

Introduction

Aflatoxin contamination of groundnut before and/or after harvest is a serious problemin most groundnut-growing areas. Preharvest aflatoxin contamination is most importantin the semi-arid tropics where drought-stress is of common occurrence (1). Drought­stress and pod-zone soil temperatures influence seed infection by Aspergillusjlavus andsubsequent aflatoxin contamination; soil temperatures in the range of 25.7-31.3C areconducive to aflatoxin contamination (2). At ICRISAT Center, we have observed aflatoxincontamination in apparently undamaged, mature seeds of drought-stressed groundnutsgrown in rainy seasons. Limited information is available on aflatoxin contamination ingroundnuts grown in postrainy seasons which are characterized by high soil temperaturesduring the pod-filling phase. In the postrainy season at ICRISAT Center, pod-zone soiltemperatures during pod development and maturation normally exceed the reportedmean upper limit (31.3C) for aflatoxin development. This paper reports results ofinvestigations into preharvest aflatoxin contamination in groundnuts exposed to terminaldrought-stress in two postrainy seasons at ICRISAT Center.

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Materials and methods

In the 1990/91 postrainy season, seed samples were collected from two field trials - (i)81 genotypes subjected to continuous terminal drought-stress (Experiment I), and (ii)49 genotypes grown under a water-deficit gradient (Experiment II). Tests for aflatoxincontamination and A.flavus infection were performed on pooled seeds of genotypes inorder to obtain a few large seed samples since genotypic differences in toxin productionwere not aimed at.

In the 1991/92 postrainy season, field trials were conducted with four selectedgenotypes, susceptible to preharvest seed infection by A. flavus, which were subjectedto terminal drought stress conditions (Experiment 111).

All trials were conducted on Alfisols at ICRISAT Center, Patancheru, India. Alfisolfields had 73-80% sand, 6-8% silt and 14-18% clay with an available water-holdingcapacity of90-110 rom.

Experiment IEighty-one genotypes were grown in 2 rows of 4 m length in a triple lattice design. Therows were 60 em apart and seeds were sown singly at lO-cm spacing along the rows.The trial was irrigated at 10-day intervals until 90 days after sowing (DAS) and droughtstress was then imposed by withholding irrigation. Genotypes were harvested 135 DAS,and plants windrow-dried for 2 days. Mature pods were picked from the plants andshade-dried to a seed moisture content of7-8%.

Experiment IIForty-nine genotypes were grown in a randomized block design in three replications.The genotypes were each sown in two rows across eight broadbeds (beds 1.2 m wide),the rows being 30 cm apart, and seeds were sown singly at 10-cm spacing along therows. The crop received uniform irrigation at 7-day intervals up to 90 DAS, after whichtime the line-source sprinkler irrigation system (3) was used to create a gradient ofwater-deficit from beds 1 to 8. The drought intensity (percent water deficit) in each bedwas determined using the method described by Mehan et al. (4). The methods ofharvesting and produce drying were the same as described for experiment I.

Experiment IIIIn the 1991/92 postrainy season, the genotypes JL 24 and EC 76446(292) in one trialand Robut 33-1 and ICGS 11 in another trial, were grown in a randomized block designwith three replications. Each genotype was sown at lO-cm spacing in 20 rows of 9 mlong and 60 em apart. The trials were irrigated at lO-day intervals until 90 DAS anddrought stress was then imposed by withholding irrigation. Genotypes were harvested10 days before harvest (125 DAS) and at normal harvest (135 DAS), and mature podswere hand-picked.

Pod-zone soiltemperaturesFor Experiments I and III, copper-constantan thermocouples placed in the pod-zone (at5 em depth) recorded minimum and maximum soil temperatures at 0600 and 1400 h onalternate days during the drought-treatment period.

Aflatoxin analysisInExperiment I, three samples-sets (A, B, and C) were obtained by grouping 81 genotypesat random into three sets of 27 each in all three replicates. For each set, pods were hand-

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shelled and 100-g seeds of all 27 genotypes were pooled and segregated into (a) apparentlyundamaged seeds and (b) damaged/discolored seeds. Damaged seeds included seedswith mechanical damage and insect-damaged seeds. Discolored seeds included seedsmainly with Macrophomina phaseolina infection. These damaged and discolored seedsare hereafter referred to as damaged seeds. Sample lots of undamaged seeds (l kg) anddamaged seeds (200 g) were finely ground, and two 50-g sub-samples were tested foraflatoxin content by the method of Pons' et al. (5).

For Experiment II, 100 g seed of all 49 genotypes were pooled for each bed (2, 4,6 and 8) in all three replicates. Each pooled seed-lot was segregated into (a) undamagedand (b) damaged/discolored categories, and two 50-g sub-samples were analyzed foraflatoxins.

For Experiment III, each replicated sample was segregated into undamaged anddamaged/discolored seeds and 50-g seed samples were tested for aflatoxin content.

Seed infection by Aspergillus ttevusAspergillus flavus seed infection levels were determined for pooled seeds ofall samplesin the experiments I and II using the method of Mehan et al. (4); 100 seeds were usedfrom undamaged seed-lots, and 25 from damaged seed-lots. Two hundred seeds wereexamined for each of the samples from Experiment III.

Results

Experiment IMinimum and maximum pod-zone soil temperatures during the drought treatment periodvaried from 15 to 26.5C and 34.3 to 43.9C respectively, in the 1990/91 postrainy season.

High levels of aflatoxin BI

were found in damaged seed-lots of all three sample-setswith mean concentrations ranging from 1480 to 2467 ug/kg (Table 1). Aflatoxincontamination was negligible in all undamaged seed-lots; concentrations of only 1-2ug/kg were detected in some samples. Levels of A. flavus seed infection ranged from22 to 26% in undamaged, and from 45 to 65% in damaged seeds in three sample sets.

Experiment IIWater deficits of 25.3%,4 I .3%,68.3%, and 84.3% were recorded in beds 2, 4, 6 and 8,respectively. Aflatoxin B. was detected only in damaged seeds (Table 2). Toxinconcentration increased with increasing water deficit, but aflatoxin levels were varia­ble, particularly among replicate samples from the greater water deficits conditions.Aflatoxin was not detected in any apparently undamaged seed samples. Levels of A.flavus seed infection ranged from 15 to 20% in undamaged, and from 34 to 52% indamaged seeds.

Experiment IIIMinimum and maximum pod-zone soil temperatures during the drought treatment periodranged from 17.3 to 29.4C and 32.9 to 49.3C respectively. Minimum temperatures werebetween 25 and 29.4C for the last 12 days of the drought treatment.

Aflatoxin B1 was found in damaged samples of all four genotypes both before and atharvest (Table 3). Seed samples of JL 24 and ICGS 11had significantly higher aflatoxincontamination than those of two other genotypes before harvest. No significant differencesin levels of aflatoxin occurred among genotypes at harvest. No toxin was detected inany apparently undamaged seed-lots of the four genotypes at either harvest time.

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Tab 1 - Aflatoxin contamination In apparently undamaged and damaged seeds ofgroundnut crop subjected to late-season drought stress In the 1990191 postralnyseason at ICRISAT Center

Mean aflatoxin B, (J.1g1kg)Sample-set'

Seed condition

Apparently undamaged

A

0.3(0-1)'

B

1.3(0-2)

C

0.3(0-1)

Damaged

SE

1480(440-2400) 2467(2200-2700) 2000(1600-2200)(3.08:0.34)3 (3.39:0.05) (3.30:0.05)

(:to.13)

'Each sample-set consisted of seeds from 27 genotypes.2Figures in parentheses are ranges over replications.3Log10transformed values: SE.

Tab 2 - Aflatoxin contamination In apparently undamaged and damaged seeds ofgroundnut crop subjected to different water-deficits for 30 days before harvest Inthe 1990191 postralny season

Aflatoxin (J.1g1kg)% water deficit

25.3 41.3 68.3 84.3Seed condition (Bed 2) (Bed 4) (Bed 6) (Bed 8)

Apparently undamaged 0 0 0 0

Damaged 26(21-31)1 121(13-250) 268(105-450) 850(100-2250)(1.42:0.08)2 (1.85:0.54) (2.36:0.29) (2.55:0.60)

SE (:0.33)

1Figuresin parentheses are ranges over replications.2Log transformed values: SE.

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Tab 3 - Aflatoxin contamination In apparently healthy and damaged/discolored/moldy seeds of groundnut genotypes sUbjected to late-season drought stress Inthe 1991/92postralny season at ICRISATCenter

Aflatoxin 81 (Ilg/kg)

Before harvest At harvest

Undamaged UndamagedGenotypes seeds Damaged seeds seeds Damaged seeds

JL24 0 2000.0 (3.15)1 0 125.0(2.09)

EC 76446 (292) 0 1.3 (0.32) 0 450.0(2.53)

ices 11 0 318.0 (2.03) 0 13.3(1.14)

Kadiri 3 0 2.4 (0.51) 0 89.3(1.55)

SE (±0.36) (±0.42)

lFigures in parentheses are log transformed values.

Aspergillus flavus infectionwas frequent in both apparently undamaged and damagedseeds of thegenotypes bothbefore andatharvest. Levels ofseed infection in thegenotypesrangedfrom 17.0to 26.8%.

Discussion

The present investigations have clearly demonstrated that apparently undamagedgroundnuts producedunderimposed droughtstressin thepostrainy seasonat ICRISATCenter were free from, or had only negligible aflatoxin contamination. During thepostrainy seasons, maximum soiltemperatures ranged from 32.9to49.3Cand minimumtemperatures from 15 to 26.5Cunderterminaldroughtconditions. These temperaturesareunlikely to favoraflatoxin production according to Blankenship et al. (6,7)andColeet al. (2,8)whoreportedthat aflatoxin contamination couldnotoccureven in drought­stressed groundnuts at soil temperatures below 25.7C or above 31.3C. However,Blankenship et al. (6)andColeet al. (8)conducted theirexperiments inenvironmentallycontrolled plotsunderconstanttemperature regimes. In thepresentinvestigations, onlynegligible aflatoxin(1-2 ug/kg) wasdetectedin someundamaged seed samplesunderfluctuating temperature regimes of 15 to 49.3C.Fluctuating diurnal temperatures mayinfluence aflatoxin formation and accumulation rate. In order to elucidate pod-zonetemperatures requiredfor preharvest aflatoxin contamination underfield conditions inthesemi-arid tropics, it would benecessary tomonitordiurnal changes insoiltemperatureand moisture levels, and relate these to toxin contamination at differentstagesof podmaturity. However, the reasons for significant aflatoxin contamination of onlydamagedseedsin both postrainyseasons are not known.Perhapsaflatoxin contamination couldoccurin seedsof pods subjected to any kind of damageduringthe early stagesof theirdevelopment.

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The role of phytoalexins in resistance to aflatoxin formation in sound maturegroundnuts grown under nonstress conditions has been highlighted (9), but thismechanism of resistance is not likely to beactive under drought-stress conditions due tolow «0.95 awl water activities of seed.

Increased levels of aflatoxin in damaged seeds under greater water deficits indicatethat aflatoxin formation could also occur at temperatures above the reported optimumrange (2). However, in the present studies the diurnal changes in soil temperatures werenot monitored, and these could play an important role in induction of alfatoxin formation.High variation in aflatoxin B

1levels observed in some samples of damaged/discolored

seeds tested before harvest and at harvest can be attributed either to differences ininfections by A. flavus strains varying in toxin production abilities, or to the differingproportions of damaged and discolored seeds in samples. Sampling variations may alsocontribute to such variations as seeds were not graded based on degree of damageJdiscoloration.

Aspergillusflavus infection was observed in both apparently undamaged and damagedseeds, but significant aflatoxin formation occurred only in damaged seeds. In most cases,levels of seed infection were considerably higher in damaged than in apparentlyundamaged seeds. Possibly, greater fungal biomass in damaged seeds might explain themuch higher levels of aflatoxin observed in these seeds.

The present results indicate very low risk of preharvest aflatoxin contamination inundamaged seeds of groundnuts grown during postrainy season even under drought­stress conditions. This supports the recommendation to growers and processors toeliminate visually discolored and moldy groundnuts to reduce overall aflatoxincontamination of the crop produce. The presence of viable mycelia of A. flavus inapparently undamaged seeds also highlights the need for good storage conditions withlow temperature to prevent further growth of the toxigenic fungus/aflatoxin productionin seeds.

It would be usefu1 to validate low or no risk of preharvest aflatoxin contamination ofgroundnuts grown in residual moisture conditions in the postrainy season in differentagroecological regions which are characterized by high temperatures and drought stressduring pod-filling phase.

References

Mehan VK (1987) The aflatoxin contamination problem in groundnut: control withemphasis on host plant resistance. In: Proceedings of the First Regional GroundnutPlant Protection Group Meeting, 15-21 Feb 1987, Harare, Zimbabwe, Lilongwe,Malawi: InternationalCrops Research Institutefor the Semi-Arid Tropics (ICRISAn,Regional Groundnut Program for Southern Africa, pp 63-92

2 Cole RI, Sanders TH, Hill RA, Blankenship PD (1985) Mean geocarpospheretemperatures that induce preharvest aflatoxin contamination of peanuts under droughtstress. Mycopathologia 91:41-46

3 Hanks RI, Keller I, Rasmussen Vp, Wilson GD (1976) Line source sprinkler forcontinuous variable irrigation: crop production studies. Soil Sci Soc Am I 40:426­429

4 Mehan VK, Rao RCN, McDonald D, Williams IH (1988) Management of droughtstress to improve field screening of peanuts for resistance to Aspergillus flavus.Phytopathology 78: 659-663

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5 Pons WA, Cucullu AF, Lee LS, Franz AO, Goldblatt LA (1966) Determination ofaflatoxins in agricultural products: use of aqueous acetone for extraction. ] AssocOffic Anal Chemists 49:554-562

6 Blankenship PO, Cole RI, Sanders TH, Hill RA (1984) Effect of geocarpospheretemperature on preharvest colonization ofdrought-stressed peanuts by Aspergillusflavus and subsequent aflatoxin contamination. Mycopathologia 85: 69-74

7 Blankenship PO, Sanders TH, Domer JW,Cole RI, Mitchell BW (1989) Enginee­ring aspects of aflatoxin research in groundnuts: Evolution of an environmentalcontrol plot facility. In: McDonald 0, Mehan VK (Eds), Aflatoxin contaminationofgroundnuts: Proceedings of the International Workshop, 6-9 OCT 1987, ICRISATCenter, India. Patancheru, AP 502 324, India: ICRISAT, pp 269-278

8 Cole RI, Sanders TH, Domer JW, Blankenship PO (1989) Environmental conditionsrequired to induce preharvest aflatoxin contamination of groundnuts: Summary ofsix years' research. In: McDonald 0, Mehan VK (Eds), Aflatoxin contamination ofgroundnuts: Proceedings of the International Workshop, 6-9 OCT 1987, ICRISATCenter, India. Patancheru, AP 502 324, India: ICRISAT, pp 279-287

9 Domer JW,Cole RI, Sanders TH, Blankenship PO (1989) The interrelationship ofkernel water activity, soil temperature, maturity, and phytoalexin production inpreharvest aflatoxin contamination of drought-stressed peanuts. Mycopathologia105:117-128

Manuscript received Aug. 13, 1993; accepted Nov. 4, 1994.

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