Thirteen seed samples of lupines (Lupinus termis Forrsk.) were evaluated for their phytosanitary status
by incubation on potato-dextrose agar (PDA) and moist sterile filter papers (blotter method) at 28±2ºC. The seeds
were also assayed for the presence of aflatoxins and toxigenic fungi. Forty-seven species and 10 varieties in 14 genera
of fungi were recovered. Among these, 45 species and 10 varieties were new to lupine seeds, and 5 species and 2
varieties were new to the mycoflora of the Sudan. The genus Aspergillus (10 species, 8 varieties) was the most
common, followed by Rhizopus (1 species), Fusarium (6 species) and Alternaria (5 species), while the remaining
genera (Chaetomium, Cladosporium, Curvularia, Drechslera, Penicillium, Phoma, Emericella, Mucor, Sclerotium,
Ulocladium) displayed lower levels of contamination. Of possible pathogens on lupine plants, Alternaria (5 species)
(stem lesion) and F. oxysporum (root rot) were recovered from the seeds. Thin-layer chromatographic analysis of
chloroform extracts of the 13 seed samples revealed that two samples contained very low concentrations of aflatoxins
B1 and B2 (4.5-6.5 µg/kg)

El Nagerabi, Seifeldin Ahmed Faragalla

Elshafie, Abdulqader Elzein

Abdulqader Elzein Elshafie

Seifeldin Ahmed Faragalla El Nagerabi

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257Phytopathol. Mediterr. (2000) 39, 257-262IntroductionThe lupine (Lupinus termis Forrsk.) is an an-nual herb belonging to the tribe Genisteae (Fabace-ae). It is an important crop in unfertile soils, whereit is superior to cereals (Pate et al., 1985). In theSudan, it is grown mainly on the sandy soils inDarfur and Kordofan States (Western Sudan). Thiscrop is subject to infection from various seedbornefungi (Gärber and Jahn, 1990). Some of thesemoulds secrete different types of mycotoxins in theseeds. Such toxins and especially aflatoxins causevarious mycotoxicosis to man and his domestic live-stock (Zohri and Abdel Gawad, 1992). In the Su-Corresponding author: El-Nagerabi S.A.F.Fax: + 967 4 203101E-mail: nagerabi@hotmail.comComposition of mycoflora and aflatoxins in lupine seedsfrom the SudanSEIFELDIN AHMED FARAGALLA EL-NAGERABI and ABDULQADER ELZEIN ELSHAFIE1Department of Microbiology, Faculty of Science, Taiz University, The Republic of Yemen1Department of Biology, College of Science, Sultan Qaboos University, Muscat, Sultanate of OmanSummary. Thirteen seed samples of lupines (Lupinus termis Forrsk.) were evaluated for their phytosanitary statusby incubation on potato-dextrose agar (PDA) and moist sterile filter papers (blotter method) at 28±2ºC. The seedswere also assayed for the presence of aflatoxins and toxigenic fungi. Forty-seven species and 10 varieties in 14 generaof fungi were recovered. Among these, 45 species and 10 varieties were new to lupine seeds, and 5 species and 2varieties were new to the mycoflora of the Sudan. The genus Aspergillus (10 species, 8 varieties) was the mostcommon, followed by Rhizopus (1 species), Fusarium (6 species) and Alternaria (5 species), while the remaininggenera (Chaetomium, Cladosporium, Curvularia, Drechslera, Penicillium, Phoma, Emericella, Mucor, Sclerotium,Ulocladium) displayed lower levels of contamination. Of possible pathogens on lupine plants, Alternaria (5 species)(stem lesion) and F. oxysporum (root rot) were recovered from the seeds. Thin-layer chromatographic analysis ofchloroform extracts of the 13 seed samples revealed that two samples contained very low concentrations of aflatoxinsB1 and B2 (4.5-6.5 µg/kg).Key words: aflatoxins, lupine, Lupinus termis, mycoflora, pathogens.dan, with its diversity in climatic conditions, rain-fall and vegetation, large numbers of pathogenicand saprophytic fungi are expected to occur(Elshafie, 1986). However, little is known about therusts, smuts, powdery mildews and leaf spot dis-eases in this country (Tarr, 1963).Lupine production in the Sudan as that of oth-er crops, is characterized by poor sanitary practic-es, low seed production and a scarcity of research.The present study was carried out to improve thissituation.Materials and methodsCollection of seed samplesThirteen seed samples of lupine (1 kg each) wereobtained from local markets in Khartoum. Thesamples were collected randomly during the study258 Phytopathologia MediterraneaS.A.F. El-Nagerabi and A.E. Elshafieperiod and represented the harvesting seasonsfrom 1993 to 1996. The samples were stored inpolyethylene bags in refrigerator at 5ºC and weredrawn and examined in accordance with the inter-national rules of the Seed Testing Association(ISTA, 1966).Moisture content determinationFrom each sample lot, four replicates of 100seeds were ground in an electric grinder and theflour was dried at 105ºC to constant weight. Themoisture content was calculated as a percentageof the initial weight.Estimation of fungi and seed germination assaysEight-hundred seeds from each sample weresurface disinfected with mercuric chloride (0.1%in water) for 5 min, washed in several changes ofsterile distilled water and incubated on potato-dex-trose agar (PDA) or on sterile filter paper (blottermethod) at 28±2ºC. In each plate, eight seeds werespaced according to their size. Ten days later, thecolonies of fungi, which had developed around theseeds, were examined and identified. The percent-age of infected seeds and all fungal species isolat-ed were recorded.Aflatoxins assaysThe bright greenish-yellow fluorescence meth-od (BGYF) (Bothast and Hasseltine, 1975) andthin-layer chromatography (TLC) of chloroformextracts from seeds (Singh et al., 1991) were usedfor detection of aflatoxins in the 13 seed samplesof lupine. For aflatoxins extraction, 500 g from eachsample were powdered, 50 g were added to 250 mlof ethanol:water (60:40 v/v) and shaken on a me-chanical shaker for 30 minutes. The solution wasleft to sediment, filtered and 125 ml of the filtratewere placed into a 250 ml separating funnel towhich 30 ml of saturated sodium chloride and 50ml hexane were added. The solution was vigorous-ly shaken for 2 minutes and left to separate. Thelower methanol water layer was collected into an-other 250 ml separating funnel, 50 ml of chloro-form were added and the mixture shaken with fre-quent venting to reduce pressure from vapourisedchloroform. The chloroform layer was drained intoa conical flask containing 5 g of cupric carbonate,shaken, left to settle, filtered through Whatmanfilter paper No. 42 having a bed of anhydrous sodi-um sulphate and the chloroform extract was col-lected into a beaker. The cupric carbonate residuewas washed with 25 ml chloroform again and fil-tered through a sodium sulphate bed. The two chlo-roform extracts were combined, evaporated in wa-ter bath until dry and the residue was dissolved in1 ml of chloroform and transferred into a screwcap vial. The extract was kept for qualitative andquantitative estimation of aflatoxins.ResultsEstimation of fungi and seed germination assaysThe fungal species isolated from lupine seedsare listed in Table 1. Forty-seven fungal speciesand 10 varieties in 14 genera were recorded as seed-borne fungi. Forty-five species and 10 varietieswere new records for lupine seeds, and 5 speciesand 2 varieties are reported for the first time inthe Sudan. The genus Aspergillus was predomi-nant, yielding 10 species and 8 varieties (42% ofthe total fungal colony count). This genus was fol-lowed by Rhizopus (one species, 35%), Fusarium(6 species) and Alternaria (5 species) (both 7%),while the remaining 10 genera displayed very lowlevels of infection (16%). Many saprophytes andsome of the potential pathogens of lupine plantswere isolated from the seeds, which were highlycontaminated (12.75-59.75%) and displayed vary-ing levels of germination (62.0-99.5%, average82.15%) (Table 2).Toxigenic fungi and aflatoxin assaysNumerous species of toxin-producing fungi ofthe genera Aspergillus, Fusarium and Penicilliumwere recovered from the seeds of this crop. TLC ofchloroform extracts of the 13 seed samples (Table2) showed that 2 samples were naturally contami-nated with aflatoxins B1 and B2, at concentrationsranging from 4.5 to 6.5 µg/kg of seeds.DiscussionVarious authors have isolated many fungi fromthe seeds of lupine in diverse climatic regions (Hi-tokoto et al., 1981; Abdel-Hafez and Shoreit, 1986;El-Maraghy, 1989). In the present study, 47 spe-cies and 10 varieties in 14 genera were found tohave contaminated the lupine crop. In previousmycological studies on lupine and other legumes:259Vol. 39, No. 2, August 2000Mycoflora and aflatoxins in lupine seedsTable 1. Infection counts of fungi in lupine seeds tested by the blotter method and on potato-dextrose agar.Recovery of fungiType of from lupine seeds (%) Recovery Occurrencec                  Fungus recorda (average) NICbremarkFrom dishes From dishes with (%)with PDA wet filter paperAlternaria alternata A 1.00 2.50 1.75 8 MA. brassicicola A 0.00 1.25 0.63 2 RA. chlamydospora AB 0.00 1.25 0.63 3 RA. tenuis A 1.33 0.00 0.67 5 LA. tenuissima A 0.00 3.25 1.63 3 RAspergillus sp. B 0.38 1.00 0.69 6 LA. carbonarious AB 0.00 2.25 1.13 3 RA. flavus var. columnaris AB 9.63 4.90 7.27 14 HA. flavus var. flavus A 5.88 5.92 5.90 20 HA. fumigans A 0.25 1.13 0.69 5 LA. japonicus var. aculeatus AC 0.50 2.00 2.25 4 LA. japonicus var. japonicus AC 3.75 0.50 2.13 4 LA. nidulellus var. nidulans A 1.75 2.75 2.25 6 LA. niger A 7.36 5.40 6.38 24 HA. niger var. awamori AB 0.00 0.50 0.25 2 RA. oryzae AB 1.75 1.13 1.44 10 MA. parasiticus AB 0.75 0.00 0.38 3 RA. terreus var. aureus AB 2.00 1.25 1.75 6 LA. terreus var. terreus A 1.25 0.00 0.63 7 MA. violaceo-brunneus AB 0.75 0.00 0.38 2 RChaetomium sp. B 1.25 0.38 0.82 3 RC. elatum AB 0.75 0.00 0.38 2 RC. globosum A 0.88 0.00 0.44 6 LC. spirale AB 1.25 2.63 1.94 7 MCladosporium herbarum B 0.00 1.13 0.57 2 RC. oxysporum AB 1.13 1.75 1.44 11 MC. sphaerospermum A 0.38 1.00 0.69 7 MCurvularia eragrostidis A 1.25 1.00 0.63 4 LC. intermedia A 1.00 0.63 0.82 4 LC. lunata A 0.94 0.00 0.47 9 MC. lunata var. aeria A 1.00 0.75 0.88 4 LC. pallescens A 0.50 0.00 0.25 1 RC. senegalensis A 0.00 2.00 1.00 1 RDrechslera australiensis A 0.00 1.25 0.63 2 RD. ellissi A 1.00 1.38 1.19 8 MD. hawaiensis A 0.00 1.25 0.63 3 RD. spicifera A 0.88 2.50 1.69 6 LEmericella nidulans    var. nidulans A 1.75 2.75 2.25 6 LE. violacea AB 0.75 0.00 0.38 2 RFusarium sp. B 1.88 0.25 1.07 5 LF. chlamydosporum AC 0.00 7.00 3.50 2 RF. compactum AC 1.50 1.00 1.25 3 RF. oxysporum B 0.00 0.75 0.38 3 RF. graminearum AC 0.75 0.00 0.38 1 R(continued on the next page)260 Phytopathologia MediterraneaS.A.F. El-Nagerabi and A.E. ElshafieRecovery of fungiType of from lupine seeds (%) Recovery Occurrencec                  Fungus recorda (average) NICbremarkFrom dishes From dishes with (%)with PDA wet filter paperF. poae AB 2.00 1.50 1.75 4 LF. semitectum A 0.63 1.25 0.94 6 LMucor sp. C 2.50 0.00 1.25 3 RM. hiemalis A 0.00 4.00 2.00 2 RPenicillium sp. B 1.00 0.75 0.88 7 MP. citrinum A 0.75 0.00 0.38 1 RP. oxalicum A 0.75 0.00 0.38 1 RP. purpurogenum AB 0.00 2.00 1.00 3 RPhoma sp. B 1.08 0.00 0.54 4 LP. herbarum A 0.00 2.00 1.00 2 RP. sorghina A 0.00 3.00 1.50 2 RRhizopus sp. B 1.02 0.00 5.13 5 LR. stolonifer A 15.50 38.88 22.19 16 HSclerotium bataticola A 0.00 2.75 1.38 3 RUlocladium atrum A 0.00 2.50 1.25 2 RSterile mycelia     (hyaline and dark) B 0.88 1.13 1.01 7 Ma A, new record for lupine seeds; B, previously reported in the Sudan on other crops than lupine; C, new record for the Sudan.b NIC: number of isolation cases, out of 26 replicates.c H-high, more than 13 replicates, out of 26; M-moderate, between 7-13 replicates, out of 26; L- low, between 4-6 replicates out of 26;R- rare, less than 4 replicates,  out of 26.Table 2. Seed germination, fungal contamination, moisture content and aflatoxin presence (µg/kg) in 13 seed sam-ples of lupine.Sample Germination Contamination Moisture Presence/absenceNumber (%) by fungi (%) content (%) of aflatoxins1 62.00 38.00 8.73 _2 83.50 43.25 7.45 _3 86.75 55.50 13.50 _4 78.75 23.00 6.63 _5 94.00 32.50 10.04 _6 99.50 39.75 11.15 _7 69.25 42.75 8.16 B1 and B2 (4.5 µg/kg)8 85.75 36.50 7.20 _9 92.50 22.25 12.34 _10 66.00 12.75 5.42 _11 90.00 19.25 9.64 _12 70.00 59.75 8.04 B1 and B2 (6.5 µg/kg)13 90.50 42.50 12.09 _Table 1. (continued from the preceding page)261Vol. 39, No. 2, August 2000Mycoflora and aflatoxins in lupine seedsbean, cowpea, faba bean, lentil, pea and soybean,the genus Aspergillus was found to be the mostcommon (Abdel-Hafez and Shoreit, 1986; Zohri andAbdel Gawad, 1992; El-Kady and Youssef, 1993;Moslem and Parvez, 1993). In the current study,the genus Aspergillus was also the predominantgenus (10 species and 8 varieties, 42% of the totalfungal colony count), followed by Rhizopus, Fusar-ium, and Alternaria. The remaining genera dis-played very low levels of infection (16%).In a previous study (Moslem and Parvez, 1993),it was reported that infection of legume seeds withfungal genera such as Alternaria, Aspergillus,Fusarium, Penicillium, Curvularia, Drechslera,Ulocladium, Chaetomium, Mucor and Rhizopusaffected seed germinability to varying degrees. Inthe present study, the seeds of lupine were like-wise highly contaminated with various species ofall these genera (12.75-59.75%, average 35.21%)and showed much reduced seed germination (62.0-99.5%, average 82.15%).A number of serious pathogens have been re-ported in lupine seeds with varying rates of infec-tion (Werner 1991/92; Moslem and Parvez, 1993).In the current study, 5 species of Alternaria (A.alternata 1.75%, A. brassicicola 0.63%, A. chlamy-dospora 0.63%, A. tenuis 0.67%, A. tenuissima1.63%), and F. oxysporum (0.38%) were isolatedtogether with several pathogenic species from gen-era commonly known to be saprophytic genera (As-pergillus, Chaetomium, Curvularia, Drechslera,Mucor, Penicillium, Rhizopus and Ulocladium).Pulses are liable to contamination with variousmycotoxins, mainly aflatoxins (Habish, 1972; FAO,1979; EL-Maraghy, 1989). In the present study, thecommon aflatoxin producers A. flavus var. colum-naris (7.27%), A. flavus var. flavus (5.90%) and A.parasiticus (0.38%) were recovered from lupineseeds. The BGYF of 13 seed samples (Table 2) re-vealed that 2 samples contained aflatoxins. TLCof the chloroform extracts showed that these sam-ples contained low concentrations of aflatoxins B1and B2  (4.5-6.5 µg/kg seeds). It should be notedthat TLC, the type of analysis performed, is not avery sensitive technique; moreover, lupine seedswere shown to be colonized by other potential tox-igenic fungi as well. The possibility cannot there-fore be excluded that other samples were contam-inated by aflatoxins or other mycotoxins.The study performed also found that the seedsof lupine may carry serious pathogenic fungi; theseeds used for sowing should therefore be treatedchemically, stored properly. Moreover, as a safeguardto human health, all the seeds should be evaluatedearly for the presence of these toxic metabolites.AcknowledgementsThis research was supported by Prof. E.M.M.Abdel Bari of the University of Qatar. The facili-ties and space provided by the Department of Bot-any and the Computer Centre are highly appreci-ated. Thanks are due to Dr. A.H. Abdalla, Dr. S.A.Ibrahim, Dr. S. Eltigani, Dr. A.H.M. Ahmed , Miss.A.A. Behairi and Mrs. M.J.S. Dafaalla for theiradvice and encouragement.Literature citedAbdel-Hafez A.I.I. and A.A.M. Shoreit, 1986. Mycoflora ofbean, lupine and pea seeds in Egypt.  Aswan Scientificand Technical Bulletin, 7, 121-141.Bothast R.J. and C.W. Hesseltine, 1975. Bright greenish-yellow fluorescence and aflatoxin in agricultural com-modities. Applied Microbiology, 30, 337-338.El-Kady I.A. and M.S. Youssef, 1993. Survey of mycofloraand mycotoxins in Egyptian soybean seeds. Journal ofBasic Microbiology, 33(6), 371-378.El-Maraghy S.S.M., 1989. The fungal flora and natural oc-currence of mycotoxins of some leguminous varietiesand hybrid seeds.  Bulletin of Faculty of Science, AssuitUniversity, 18, 63-75.Elshafie A.E., 1986. Taxonomic studies on seed-borne fun-gi of the Sudan, II: Curvularia. Sudan Journal of Sci-ence, II, 51-70.FAO, 1979. Perspective on mycotoxins. FAO and NutritionPaper No. 13, FAO, Rome, Italy.Gärber J. and M. Jahn, 1990.  Investigation on the occur-rence of seed-borne pathogens in legumes. Nachrich-tenblatt für den Pflanzenschutz in der DDR, 44(3), 50-52.Habish H.A., 1972. Aflatoxin in haricot bean and otherpulses. Experimental Agriculture, 8, 135-137.Hitokoto H., S. Morozumi, T. Wauke, S. Sakal, and H. Ku-rata, 1981. Fungal contamination and mycotoxin-pro-ducing potential of dried beans.  Mycopathologia, 73,33-38.ISTA, 1966. International rules for seed testing.  Seed Sci-ence and Technology, 4, 3-49.Moslem M.A. and S. Parvez, 1993. Seed-borne fungi of Lensesculentus, Hordeum vulgare and Triticum aestivumfrom Saudi Arabia. International Journal of TropicalPlant Diseases, 11(1), 99-105.Neergaard P., 1977. Seed Pathology. The Macmillian PressLtd. London, UK, 3, 519-839.262 Phytopathologia MediterraneaS.A.F. El-Nagerabi and A.E. ElshafiePate J.S., W. Williams and P. Farrington, 1985. Lupine (Lu-pinus spp.). In: Grain Legume Crops.  (SummerfieldR.J., Roberts E.H. eds). William Collins, London, UK,699-746.Singh K., J.C. Frisval, U. Thrane and S.M. Mathur, 1991.An illustrated manual on identification of some seed-borne Aspergilli, Fusaria, Penicillia and their mycotox-ins. Danish Government Institute of Seed Pathology forDeveloping Countries, Hellerup, Denmark, 133 pp.Tarr S.A.J., 1963. A Supplementary List of Sudan Fungiand Plant Diseases. Mycological paper No. 85, Common-wealth Mycological Institute, Kew, Surrey, England.Werner M., 1992. Pathogenicity of eight formae specialesof Fusarium oxysporum Schlecht in relation to differ-ent plant species. Acta Mycologica, 27(1), 127-136.Zohri A.A. and K.  Abdel Gawad, 1992. Studies on mycoflo-ra and mycotoxins of cowpea cultivars. Korean Journalof Mycology, 20(3), 252-258.Accepted for publication: March 3, 2000

Composition of Mycoflora and Aflatoxins in Lupine Seeds from the Sudan

Firenze University Press: E-Journals

257
Phytopathol. Mediterr. (2000) 39, 257-262
Introduction
The lupine (Lupinus termis Forrsk.) is an an-
nual herb belonging to the tribe Genisteae (Fabace-
ae). It is an important crop in unfertile soils, where
it is superior to cereals (Pate et al., 1985). In the
Sudan, it is grown mainly on the sandy soils in
Darfur and Kordofan States (Western Sudan). This
crop is subject to infection from various seedborne
fungi (Gärber and Jahn, 1990). Some of these
moulds secrete different types of mycotoxins in the
seeds. Such toxins and especially aflatoxins cause
various mycotoxicosis to man and his domestic live-
stock (Zohri and Abdel Gawad, 1992). In the Su-
Corresponding author: El-Nagerabi S.A.F.
Fax: + 967 4 203101
E-mail: nagerabi@hotmail.com
Composition of mycoflora and aflatoxins in lupine seeds
from the Sudan
SEIFELDIN AHMED FARAGALLA EL-NAGERABI and ABDULQADER ELZEIN ELSHAFIE1
Department of Microbiology, Faculty of Science, Taiz University, The Republic of Yemen
1Department of Biology, College of Science, Sultan Qaboos University, Muscat, Sultanate of Oman
Summary. Thirteen seed samples of lupines (Lupinus termis Forrsk.) were evaluated for their phytosanitary status
by incubation on potato-dextrose agar (PDA) and moist sterile filter papers (blotter method) at 28±2ºC. The seeds
were also assayed for the presence of aflatoxins and toxigenic fungi. Forty-seven species and 10 varieties in 14 genera
of fungi were recovered. Among these, 45 species and 10 varieties were new to lupine seeds, and 5 species and 2
varieties were new to the mycoflora of the Sudan. The genus Aspergillus (10 species, 8 varieties) was the most
common, followed by Rhizopus (1 species), Fusarium (6 species) and Alternaria (5 species), while the remaining
genera (Chaetomium, Cladosporium, Curvularia, Drechslera, Penicillium, Phoma, Emericella, Mucor, Sclerotium,
Ulocladium) displayed lower levels of contamination. Of possible pathogens on lupine plants, Alternaria (5 species)
(stem lesion) and F. oxysporum (root rot) were recovered from the seeds. Thin-layer chromatographic analysis of
chloroform extracts of the 13 seed samples revealed that two samples contained very low concentrations of aflatoxins
B1 and B2 (4.5-6.5 µg/kg).
Key words: aflatoxins, lupine, Lupinus termis, mycoflora, pathogens.
dan, with its diversity in climatic conditions, rain-
fall and vegetation, large numbers of pathogenic
and saprophytic fungi are expected to occur
(Elshafie, 1986). However, little is known about the
rusts, smuts, powdery mildews and leaf spot dis-
eases in this country (Tarr, 1963).
Lupine production in the Sudan as that of oth-
er crops, is characterized by poor sanitary practic-
es, low seed production and a scarcity of research.
The present study was carried out to improve this
situation.
Materials and methods
Collection of seed samples
Thirteen seed samples of lupine (1 kg each) were
obtained from local markets in Khartoum. The
samples were collected randomly during the study
258 Phytopathologia Mediterranea
S.A.F. El-Nagerabi and A.E. Elshafie
period and represented the harvesting seasons
from 1993 to 1996. The samples were stored in
polyethylene bags in refrigerator at 5ºC and were
drawn and examined in accordance with the inter-
national rules of the Seed Testing Association
(ISTA, 1966).
Moisture content determination
From each sample lot, four replicates of 100
seeds were ground in an electric grinder and the
flour was dried at 105ºC to constant weight. The
moisture content was calculated as a percentage
of the initial weight.
Estimation of fungi and seed germination assays
Eight-hundred seeds from each sample were
surface disinfected with mercuric chloride (0.1%
in water) for 5 min, washed in several changes of
sterile distilled water and incubated on potato-dex-
trose agar (PDA) or on sterile filter paper (blotter
method) at 28±2ºC. In each plate, eight seeds were
spaced according to their size. Ten days later, the
colonies of fungi, which had developed around the
seeds, were examined and identified. The percent-
age of infected seeds and all fungal species isolat-
ed were recorded.
Aflatoxins assays
The bright greenish-yellow fluorescence meth-
od (BGYF) (Bothast and Hasseltine, 1975) and
thin-layer chromatography (TLC) of chloroform
extracts from seeds (Singh et al., 1991) were used
for detection of aflatoxins in the 13 seed samples
of lupine. For aflatoxins extraction, 500 g from each
sample were powdered, 50 g were added to 250 ml
of ethanol:water (60:40 v/v) and shaken on a me-
chanical shaker for 30 minutes. The solution was
left to sediment, filtered and 125 ml of the filtrate
were placed into a 250 ml separating funnel to
which 30 ml of saturated sodium chloride and 50
ml hexane were added. The solution was vigorous-
ly shaken for 2 minutes and left to separate. The
lower methanol water layer was collected into an-
other 250 ml separating funnel, 50 ml of chloro-
form were added and the mixture shaken with fre-
quent venting to reduce pressure from vapourised
chloroform. The chloroform layer was drained into
a conical flask containing 5 g of cupric carbonate,
shaken, left to settle, filtered through Whatman
filter paper No. 42 having a bed of anhydrous sodi-
um sulphate and the chloroform extract was col-
lected into a beaker. The cupric carbonate residue
was washed with 25 ml chloroform again and fil-
tered through a sodium sulphate bed. The two chlo-
roform extracts were combined, evaporated in wa-
ter bath until dry and the residue was dissolved in
1 ml of chloroform and transferred into a screw
cap vial. The extract was kept for qualitative and
quantitative estimation of aflatoxins.
Results
Estimation of fungi and seed germination assays
The fungal species isolated from lupine seeds
are listed in Table 1. Forty-seven fungal species
and 10 varieties in 14 genera were recorded as seed-
borne fungi. Forty-five species and 10 varieties
were new records for lupine seeds, and 5 species
and 2 varieties are reported for the first time in
the Sudan. The genus Aspergillus was predomi-
nant, yielding 10 species and 8 varieties (42% of
the total fungal colony count). This genus was fol-
lowed by Rhizopus (one species, 35%), Fusarium
(6 species) and Alternaria (5 species) (both 7%),
while the remaining 10 genera displayed very low
levels of infection (16%). Many saprophytes and
some of the potential pathogens of lupine plants
were isolated from the seeds, which were highly
contaminated (12.75-59.75%) and displayed vary-
ing levels of germination (62.0-99.5%, average
82.15%) (Table 2).
Toxigenic fungi and aflatoxin assays
Numerous species of toxin-producing fungi of
the genera Aspergillus, Fusarium and Penicillium
were recovered from the seeds of this crop. TLC of
chloroform extracts of the 13 seed samples (Table
2) showed that 2 samples were naturally contami-
nated with aflatoxins B1 and B2, at concentrations
ranging from 4.5 to 6.5 µg/kg of seeds.
Discussion
Various authors have isolated many fungi from
the seeds of lupine in diverse climatic regions (Hi-
tokoto et al., 1981; Abdel-Hafez and Shoreit, 1986;
El-Maraghy, 1989). In the present study, 47 spe-
cies and 10 varieties in 14 genera were found to
have contaminated the lupine crop. In previous
mycological studies on lupine and other legumes:
259Vol. 39, No. 2, August 2000
Mycoflora and aflatoxins in lupine seeds
Table 1. Infection counts of fungi in lupine seeds tested by the blotter method and on potato-dextrose agar.
Recovery of fungi
Type of from lupine seeds (%) Recovery Occurrencec                  Fungus recorda (average) NIC
b
remarkFrom dishes From dishes with (%)
with PDA wet filter paper
Alternaria alternata A 1.00 2.50 1.75 8 M
A. brassicicola A 0.00 1.25 0.63 2 R
A. chlamydospora AB 0.00 1.25 0.63 3 R
A. tenuis A 1.33 0.00 0.67 5 L
A. tenuissima A 0.00 3.25 1.63 3 R
Aspergillus sp. B 0.38 1.00 0.69 6 L
A. carbonarious AB 0.00 2.25 1.13 3 R
A. flavus var. columnaris AB 9.63 4.90 7.27 14 H
A. flavus var. flavus A 5.88 5.92 5.90 20 H
A. fumigans A 0.25 1.13 0.69 5 L
A. japonicus var. aculeatus AC 0.50 2.00 2.25 4 L
A. japonicus var. japonicus AC 3.75 0.50 2.13 4 L
A. nidulellus var. nidulans A 1.75 2.75 2.25 6 L
A. niger A 7.36 5.40 6.38 24 H
A. niger var. awamori AB 0.00 0.50 0.25 2 R
A. oryzae AB 1.75 1.13 1.44 10 M
A. parasiticus AB 0.75 0.00 0.38 3 R
A. terreus var. aureus AB 2.00 1.25 1.75 6 L
A. terreus var. terreus A 1.25 0.00 0.63 7 M
A. violaceo-brunneus AB 0.75 0.00 0.38 2 R
Chaetomium sp. B 1.25 0.38 0.82 3 R
C. elatum AB 0.75 0.00 0.38 2 R
C. globosum A 0.88 0.00 0.44 6 L
C. spirale AB 1.25 2.63 1.94 7 M
Cladosporium herbarum B 0.00 1.13 0.57 2 R
C. oxysporum AB 1.13 1.75 1.44 11 M
C. sphaerospermum A 0.38 1.00 0.69 7 M
Curvularia eragrostidis A 1.25 1.00 0.63 4 L
C. intermedia A 1.00 0.63 0.82 4 L
C. lunata A 0.94 0.00 0.47 9 M
C. lunata var. aeria A 1.00 0.75 0.88 4 L
C. pallescens A 0.50 0.00 0.25 1 R
C. senegalensis A 0.00 2.00 1.00 1 R
Drechslera australiensis A 0.00 1.25 0.63 2 R
D. ellissi A 1.00 1.38 1.19 8 M
D. hawaiensis A 0.00 1.25 0.63 3 R
D. spicifera A 0.88 2.50 1.69 6 L
Emericella nidulans
    var. nidulans A 1.75 2.75 2.25 6 L
E. violacea AB 0.75 0.00 0.38 2 R
Fusarium sp. B 1.88 0.25 1.07 5 L
F. chlamydosporum AC 0.00 7.00 3.50 2 R
F. compactum AC 1.50 1.00 1.25 3 R
F. oxysporum B 0.00 0.75 0.38 3 R
F. graminearum AC 0.75 0.00 0.38 1 R
(continued on the next page)
260 Phytopathologia Mediterranea
S.A.F. El-Nagerabi and A.E. Elshafie
Recovery of fungi
Type of from lupine seeds (%) Recovery Occurrencec
                  Fungus recorda (average) NIC
b
remark
From dishes From dishes with (%)
with PDA wet filter paper
F. poae AB 2.00 1.50 1.75 4 L
F. semitectum A 0.63 1.25 0.94 6 L
Mucor sp. C 2.50 0.00 1.25 3 R
M. hiemalis A 0.00 4.00 2.00 2 R
Penicillium sp. B 1.00 0.75 0.88 7 M
P. citrinum A 0.75 0.00 0.38 1 R
P. oxalicum A 0.75 0.00 0.38 1 R
P. purpurogenum AB 0.00 2.00 1.00 3 R
Phoma sp. B 1.08 0.00 0.54 4 L
P. herbarum A 0.00 2.00 1.00 2 R
P. sorghina A 0.00 3.00 1.50 2 R
Rhizopus sp. B 1.02 0.00 5.13 5 L
R. stolonifer A 15.50 38.88 22.19 16 H
Sclerotium bataticola A 0.00 2.75 1.38 3 R
Ulocladium atrum A 0.00 2.50 1.25 2 R
Sterile mycelia
     (hyaline and dark) B 0.88 1.13 1.01 7 M
a A, new record for lupine seeds; B, previously reported in the Sudan on other crops than lupine; C, new record for the Sudan.
b NIC: number of isolation cases, out of 26 replicates.
c H-high, more than 13 replicates, out of 26; M-moderate, between 7-13 replicates, out of 26; L- low, between 4-6 replicates out of 26;
R- rare, less than 4 replicates,  out of 26.
Table 2. Seed germination, fungal contamination, moisture content and aflatoxin presence (µg/kg) in 13 seed sam-
ples of lupine.
Sample Germination Contamination Moisture Presence/absence
Number (%) by fungi (%) content (%) of aflatoxins
1 62.00 38.00 8.73 _
2 83.50 43.25 7.45 _
3 86.75 55.50 13.50 _
4 78.75 23.00 6.63 _
5 94.00 32.50 10.04 _
6 99.50 39.75 11.15 _
7 69.25 42.75 8.16 B1 and B2 (4.5 µg/kg)
8 85.75 36.50 7.20 _
9 92.50 22.25 12.34 _
10 66.00 12.75 5.42 _
11 90.00 19.25 9.64 _
12 70.00 59.75 8.04 B1 and B2 (6.5 µg/kg)
13 90.50 42.50 12.09 _
Table 1. (continued from the preceding page)
261Vol. 39, No. 2, August 2000
Mycoflora and aflatoxins in lupine seeds
bean, cowpea, faba bean, lentil, pea and soybean,
the genus Aspergillus was found to be the most
common (Abdel-Hafez and Shoreit, 1986; Zohri and
Abdel Gawad, 1992; El-Kady and Youssef, 1993;
Moslem and Parvez, 1993). In the current study,
the genus Aspergillus was also the predominant
genus (10 species and 8 varieties, 42% of the total
fungal colony count), followed by Rhizopus, Fusar-
ium, and Alternaria. The remaining genera dis-
played very low levels of infection (16%).
In a previous study (Moslem and Parvez, 1993),
it was reported that infection of legume seeds with
fungal genera such as Alternaria, Aspergillus,
Fusarium, Penicillium, Curvularia, Drechslera,
Ulocladium, Chaetomium, Mucor and Rhizopus
affected seed germinability to varying degrees. In
the present study, the seeds of lupine were like-
wise highly contaminated with various species of
all these genera (12.75-59.75%, average 35.21%)
and showed much reduced seed germination (62.0-
99.5%, average 82.15%).
A number of serious pathogens have been re-
ported in lupine seeds with varying rates of infec-
tion (Werner 1991/92; Moslem and Parvez, 1993).
In the current study, 5 species of Alternaria (A.
alternata 1.75%, A. brassicicola 0.63%, A. chlamy-
dospora 0.63%, A. tenuis 0.67%, A. tenuissima
1.63%), and F. oxysporum (0.38%) were isolated
together with several pathogenic species from gen-
era commonly known to be saprophytic genera (As-
pergillus, Chaetomium, Curvularia, Drechslera,
Mucor, Penicillium, Rhizopus and Ulocladium).
Pulses are liable to contamination with various
mycotoxins, mainly aflatoxins (Habish, 1972; FAO,
1979; EL-Maraghy, 1989). In the present study, the
common aflatoxin producers A. flavus var. colum-
naris (7.27%), A. flavus var. flavus (5.90%) and A.
parasiticus (0.38%) were recovered from lupine
seeds. The BGYF of 13 seed samples (Table 2) re-
vealed that 2 samples contained aflatoxins. TLC
of the chloroform extracts showed that these sam-
ples contained low concentrations of aflatoxins B1
and B2  (4.5-6.5 µg/kg seeds). It should be noted
that TLC, the type of analysis performed, is not a
very sensitive technique; moreover, lupine seeds
were shown to be colonized by other potential tox-
igenic fungi as well. The possibility cannot there-
fore be excluded that other samples were contam-
inated by aflatoxins or other mycotoxins.
The study performed also found that the seeds
of lupine may carry serious pathogenic fungi; the
seeds used for sowing should therefore be treated
chemically, stored properly. Moreover, as a safeguard
to human health, all the seeds should be evaluated
early for the presence of these toxic metabolites.
Acknowledgements
This research was supported by Prof. E.M.M.
Abdel Bari of the University of Qatar. The facili-
ties and space provided by the Department of Bot-
any and the Computer Centre are highly appreci-
ated. Thanks are due to Dr. A.H. Abdalla, Dr. S.A.
Ibrahim, Dr. S. Eltigani, Dr. A.H.M. Ahmed , Miss.
A.A. Behairi and Mrs. M.J.S. Dafaalla for their
advice and encouragement.
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Accepted for publication: March 3, 2000


Composition of Mycoflora and Aflatoxins in Lupine Seeds from the Sudan

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