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Impact of habitats on toxigenic potential of Aspergillus flavus
0022-474X/93
J. stored Prod. Res. Vol. 29, No. 4, pp. 351-355, 1993
$6.00 + 0.00
Copyright0 1993PergamonPressLtd
Printedin GreatBritain.All rightsreserved
IMPACT OF HABITATS ON TOXIGENIC POTENTIAL OF ASPERGILLUS
FLA VU.7
K. S. BILGRAMI and A. K. CHOUDHARY University Department of Botany, Bhagalpur University, Bhagalpur 812007, India (Received far publication
30 March
1993)
Abstract-Toxigenic potentials of Aspergillus@vms strains isolated from different organic substrates, air and soils were evaluated. The frequency of occurrence of A. ~IULUSranged from 76% in maize to 87% (coconut and groundnut) and 92% in makhana (Euryaleferox indica.) Incidence was lowest in green gram (42%). Analysis of variance showed that the percentage incidence of A. ~CIUUSin the aerosphere of maize fields was significantly affected by season x location. In soil samples the frequency of occurrence of A. jZauus was high during the monsoons (76% in non-diara land soils, 69% in diara land soil). Among 1706 isolates of A. $mus obtained from different sources, 826 (48.4%) were found to be toxigenic. The frequency of non-toxigenic strains of A.j?mus was comparatively higher (ratio = 1.07: 1) than the toxigenic strains. Percentage incidence of the toxigenic strains of A. j&nrus was the highest (73.3%; ratio = 0.36: 1) in the soil samples. No attempt was made to differentiate A. jkuus and A. parasiticus and therefore all references to A. fkvus include A. parasiticus. Key words-Aspergillus
jfavus, substrates, soils, aerosphere.
INTRODUCTION
Aspergillus flavus Link contaminates a variety of agricultural commodities with aflatoxins. In previous studies on collections of toxigenic isolates of A. flaws from different natural substrates (Wallbridge, 1963; Austwick and Ayerst, 1963; Borut and Joffe, 1966; Rao et al., 1965; Bilgrami, 1985; Sinha, 1990) the ratio of non-toxigenic and toxigenic strains of A. flaous varied with source and location of isolations. However, there is no direct evidence suggesting a correlation between mycotoxin producing ability and distribution of an isolate in nature. Some indirect evidence suggested for example greater occurrence of cyclopiazonic acid (a secondary metabolite) in the strains of A.JEauus isolated primarily from tropical countries (Manabe and Tsuruta, 1978; Wicklow, 1983). In the present investigation strains of A.JEazw were isolated from diverse habitats viz. cereals, pulses, oilseeds, dry fruits, spices, air (aerosphere of maize fields) and soils. Ratio of the non-toxigenic and toxigenic isolates of A.$uuus in relation to different habitats were evaluated. MATERIALS
AND
METHODS
Seeds of different cereals (maize, wheat, rice, barley), pulses (Cujunus cajun-arhar, Phuseolus mungo-green gram), oilseeds (groundnut, Brussicujunceu-mustard), dry fruits (coconut, Euryule ferox indicu-makhana) and spices (coriander, cardamom and chilli) were obtained from different markets of Bhagalpur and its surrounding areas. For cereals, pulses and oilseeds, each sample comprised at least 1 kg seeds which were collected in polythene bags. The sample size of dry fruits and spices comprised 250 g each. Air and soil samples were collected from different fields of two localities, Locality A was near Sabour Agricultural College, about 8 km east of Bhagalpur University possessed generally loamy, clay-loam soil, comparatively rich in organic matter (0.42-0.8%) and alkaline (pH 7.2-8.2). Locality B, about 12 km west of the University was flood prone diara land, where Khurif crops during harvesting (July-September) remained either fully or partially submerged under flood waters. The soil of this locality was mostly sandy or sand-silt-clay with comparatively lesser organic matter (0.3-0.6%) and was also alkaline (pH 7.2-7.8). Isolation of mycoflora from different substrates was carried out using the blotter test and agar plate methods (ISTA, 1966). All plates were incubated for 7 days. Seeds were examined under the 351
352
K. S. BILGRAMI
and A. K. CHOUDHARY
Table I. Percentage frequency of A. flaws in different cereals, pulses, oilseeds and dry fruits Incidence of A. flow Substrates
Number of samples
Number with A. flmrtrs
% Gccurrence
172 140 60 42
131 58 32 23
76 42 53 55
Cereals
Maize Wheat Rice Barley
Pulses
Arhar Green gram
40 25
27 II
68 44
Oilseeds
Groundnut Mustard
15 72
I3 31
87 51
Dry fruits
Coconut Makhana
30 25
26 22
87 92
Spices
Coriander Cardamom Chilli
30 30 50
21 I9 29
70 63 58
stereo-microscope from the fourth day and the developing mycoflora isolated, purified and maintained on PDA and Czapek-Dox agar medium for further studies. Soil samples were collected from maize fields in both localities from January to September. Each month five random soil samples from each locality were taken at depths of O-3 cm. The soil dilution technique (Waksman, 1952) was used for isolation of soil mycoflora. Plates were incubated at 28 + 2°C for 7 days. After incubation, colonies were counted and the number of fungi per gram of dry weight estimated. The frequency of occurrence of A. flavus was calculated by the following formula: Frequency =
No. of colonies of A.$uvus x loo Total count
Twenty Petri plates containing Czapek-Dox agar were exposed in the fields of both localities from January to September. The duration of exposure ranged from 12-14 h covering from evening to next morning. Exposed plates were incubated (28 +2”C) for 45 days. Isolation and identification of fungi were subsequently carried out. The relative frequency of A. jhvus was noted each fortnight. Statistical analysis (analysis of variance) was performed in order to assess the seasonal incidence of A. JIauus in both the localities. The aflatoxin producing potential of A. frauus isolates was tested in SMKY liquid medium (sucrose-200 g, MgSO, . 7H20--0.5 g, KNO,-3 g, yeast extract-7 g, distilled water-l 1)(Diener and Davis, 1966). Aflatoxin was extracted in chloroform and both qualitative and quantitative analysis carried out. Qualitative detection was by TLC using toluene : isoamylalcohol : methanol (90: 32: 2 v/v/v) as solvent system (Reddy er al., 1970). Quantitative assays of aflatoxin were Table 2. Incidence of A. jidw.r in the aerosphere of maize fields at Bhagalpur (2-way table) % incidence of Aspergillw J?avus in different seasons Season
Winter (Jan-Mar) Summer (Apr-Jun) Monsoon (Jul-Sep) Mean
Locality A(L,)
Locality B(L,)
Mean
23.2 (28.5) 35.5 (32.3) 66.1 (54.6) 41.6 (39.8)
23.0 (28.1) 52.7 (46.5) 59.8 (50.8) 45.2 (41.8)
23. I (28.3) 44.1 (41.4) 63.0 (52.7) 43.4 (40.8)
Figures in parentheses indicate transformed values. Since the original values were in percentage, these were therefore, transformed into degrees and then analysis of variance was done. (Angle =arc sin Js where, L, = Non-diara land and L, = Diara land). 5% CD for season = 5.322. 5% CD for season x location = 7.526. (Note : Analysis of variance was performed in order to evaluate the effect of locality and interaction effect of season x location for the incidence of A. flows in the aerosphere of maize fields.)
Impact of habitats on toxigenic potential of A. Jlauus
353
Table 3. % Relative density of A. fIouw in the soils of Bhagalpur Relative density of A. @wus (%)
Average number of fungus/g dry wt Diara land of soils soil (Locality B) (Fungi x 10s)
Non-diara land soil (Locality A)
Seasons Winter (Jan-Mar) Summer (Apr-Jon) Monsoon (Jul-Sep)
38
54
5.2
33
21
4.8
76
69
8.6
determined spectrophotometrically (AOAC, 1984). Chemical confirmation was by trifluoroacetic acid (Stack and Pohland, 1975). In the present investigation, no attempt has been made to differentiate A. j&.ruus from A. parasiticus, so all references to A. frauus include A. parasiticus.
RESULTS
Aspergillusjlavus, A. niger, A. ochraceus, Aspergillus spp., Penicillium citrinum, other Penicillium spp., Fusarium moniliforme, F. equiseti and other Fusarium spp. were the most frequent fungi in different substrates. Other common fungi included Rhizopus stolonifer, Mucor spp., Alternaria spp., Cur&aria spp., Drechslera spp., Chaetomium spp., Lasiodiplodia theobromae and sterile mycelia. Infection rates varied from 24% (green gram) to 62.7% (maize). The frequency distribution of fungal species isolated from the various sources varied substantially. Aspergillusflauus was the dominant fungus with its frequency ranging from about 76% in maize to 87% in coconut and groundnut and 92% in makhana (Table 1). Its incidence was lowest in green gram (42%). It is clear from the analysis of variance (Table 2) that the percentage incidence of A. flaws in the aerosphere of maize fields was significantly affected by season x location. However, location alone did not have any significant effect. Maximum incidence of A.Jlavus was recorded during the monsoon season (63%) followed by summers (45%) and winters (24%). These differences were statistically significant. Crops grown in locality B (flood prone diara land) had much higher incidence of A. frauus during summers (53%) than in locality A (dry area, 36%). In the soil samples, the frequency of isolation of A. flaws was also high during the monsoons (76% in locality A, 69% in locality B) (Table 3). The lowest incidence (21%) of A. flavus was observed during the summers in locality B. Average number of fungi per gram dry weight of soils varied from 4.8 x lo8 to 8.6 x 10’. Of 1706 isolates of A. flaws obtained from different sources screened for aflatoxin production in SMKY liquid medium (Table 4), 826 (48%) isolates were found to be positive. Overall, the Table 4. Aflatoxin oroducine ootentialities of A. Aavus obtained from different habitats
A
B
C
Ratio of nontoxigenic and toxigenic isolates
240 90 130 I80 85 96 II0 185 120 60 60 60 60 110 120 I706
149 38 72 70 36 35 63 77 61 26 23 I9 24 45 28 826
62 42.2 55.4 39 42.4 36.5 57.3 41.6 50.8 43.3 38.3 31.7 40 40.9 73.33 48.4
0.61: I 1.37: I 0.81: I 1.57: I 1.36: I 1.74: I 0.75: I 1.4O:l 0.96: I 1.31:l 1.60: I 2.16: I 1.5:l l.4:1 0.36: I 1.07:l
Number of isolates Source of isolates 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Maize Wheat Rice Barley Arhar Green gram Groundnut Mustard Coconut Makhana 11.Coriander 12. Cardamom 13. Chilti 14. Aerosphere 15. Soils Total :
No. of isolates producing allatoxins B,
B, + B,
42 7 I7 15 II I2 18 34 19 4 5 5 II 14 28 242 (29.3%)
67 la 31 34 I4 I9 33 28 I7 I6
1 I2 6 8 I7 321 (39%)
B,+B,+G, 35 13 20 21 9 4 9 12 23 6 17 2 7 23 32 233 (28.2%)
B,+B,+G,+G, 5 4 2 3 3 2
II (3.&
Amount of aflatoxin (ppm) Min. I trace 2 2 trace trace 3 trace 7 5 trace 3 trace 2 a trace
Where A = Number of isolates screened, B = Toxigenic isolates, C = % toxigenic isolates, Min. = Minimum, Max. = Maximum.
Max. 21 15 18 I8 I3 18 I8 20 23 la I? I2 7 21 26 26
354
K. S. BILGRAMI and A. K. CHOUDHARY
incidence of non-toxigenic strains was higher and the ratio of non-toxigenic and toxigenic strains of A. ftavus was 1.07: 1. While considering different sources of isolation, percentage incidence of the toxigenic strains of A.Juuus was the highest (73.3%) in the soil samples, where the ratio of non-toxigenic and toxigenic isolates was 0.36: 1. The amount of aflatoxin produced by the isolates obtained from the soil was also a maximum, averaging 26 ppm in the liquid medium used. The incidence of the toxigenic isolates of A. flauus varied considerably from the different substrates. Toxigenic isolates of A.$avus ranged in cereals from 40 to 62%, in pulses 36.5 to 42.4%, in oilseeds 41.6 to 57.3%, in dry fruits 43.3 to 51%, in spices 32 to 40%, while in air it was 41% (Table 4). Carbohydrate rich materials had comparatively higher incidences of toxigenic isolates. Among the cereals, the incidence of the toxigenic isolates was highest (62%) in maize. Aflatoxin B, production of A. flavus strains isolated from different substrates was highest in the strains obtained from coconut (23 ppm) followed by maize (21 ppm). It was low by the isolates obtained from chilli (7 ppm) and cardamom (12 ppm). Toxigenic isolates of A. fEavus which were distributed in wide range of habitats, elaborated varying fractions of aflatoxin in liquid media. Aflatoxin B, was produced by all the toxigenic isolates of A.Jlavus (826), whereas, 242 (29.3%) isolates could produce aflatoxin B, only. 321 (39%) toxigenic isolates were capable of elaborating both aflatoxin B, and B,, while 233 (28.2%) isolates produced B,, B2 and G,. All the four components of aflatoxins (viz. B,, Bz, G, and G,) were produced by 30 isolates only (3.4%). None of the isolates, however, were able to elaborate aflatoxin B,, G, or Gr in absence of B,. The range of aflatoxin production in different isolates of A.flavus varied from trace to 26 ppm.
DISCUSSION A high incidence of A. fravus in the aerosphere of maize crops at and around Bhagalpur was observed during the monsoons followed by summers. Correlation between air-borne inoculum of A. fravus with aflatoxin contamination in fields has been reported earlier (Lillehoj et al., 1980; Lee et al., 1986). Crops grown in locality B had a much higher incidence of A. flavus during summers (53%) than in locality A (36%), perhaps due to extreme scarcity of water (Diener et al., 1987). Large frequency of occurrence of A.&vus were observed in the soils during the monsoons. This is in conformity with the results of Angle (1987) who reported a high incidence of A. jSzvus in the soil samples. In soils, plant residues of susceptible crops (viz. maize, peanuts) have been reported as congenial focal points for development of A. fiavus (Lillehoj, 1987). Because it is known that A. JEavus produces only B aflatoxins, it can be deduced that 233+30 = 263 (ca 30%) of the toxigenic isolates were A. parasiticus. As A. parasiticus is always toxigenic, 26311706, i.e. 15.4%, were A. parasiticus. While taking different habitats into account, it was observed that the frequency of non-toxigenic strains of A. JEavus was comparatively higher (ratio 1.07: 1) than the toxigenic ones. Considerable variations were observed in the distribution of non-toxigenic and toxigenic isolates of A. j&us on different organic substrates/air/soils. Bilgrami (1987) has suggested that the non-toxigenic forms are the wild type while the toxigenic ones are of mutant types. Mutants by and large have lower selective advantage due to increased genetic load in them, therefore, their number is lesser as compared to the non-toxigenic ones. Lemke et al. (1989) on the basis of single spore cultures of A. flavus on several succeeding generations have also confirmed it. Among different organic substrates, carbohydrate rich seeds were quite favourable abode for the selection of the toxigenic isolates of A. Jhzvus. Abdollahi and Buchanan (198 1) have suggested that maximum toxin yields are dependent on high concentrations of specific carbohydrates. It has been argued that aflatoxin biosynthesis may be regulated by a carbon-catabolic induction process, in which the built up of the key intermediates or alterations of energy status of the cell leads to the induction of aflatoxin synthesis (Buchanan and Lewis, 1984; Buchanan et al., 1985). Fatty acids and carbohydrate contents or some toxin stabilizing factor in coconut might be responsible for selection of highly toxigenic strains of A. J&zvus.
Impact of habitats on toxigenic potential of A. Jlavus
355
Acknowledgement-Authors are thankful to United States Department of Agriculture (USDA) for financial assistance out of U.S.-India Aid Programme (Project No-IN-ARS-253: Grant No. F.G. In-642). REFERENCES Abdollahi A. and Buchanan R. (1981) Regulation of aflatoxin biosynthesis: characterization of glucose as an apparent inducer of aflatoxin production. J. Food Sci. 46, 141-146. Angle J. S. (1987) Aflatoxin and aflatoxin producing fungi in soil. In AJuroxin in Maize, Proceedings of the Workshop (Edited by Zuber M. S., Lillehoj E. B. and Renfro B. L.), pp. 152-163. CIMMYT, Mexico. AOAC (1984) Natural poisons. In Qficiul Methods of Analysis (Edited by Stoloff L. and Scott P. M.), pp. 477-500. Association of Official Analytical Chemists, Washington DC. Austwick P. K. C. and Averst G. (1963), Toxic uroducts in groundnuts. Groundnut microflora and toxicitv. Chem. Ind. 1963, 55561. Bilgrami K. S. (1985) Mycotoxins in dry fruits and spices. Final Technical Report of UGC Project, Bhagalpur University, p. 80. Bilgrami K. S. (1987) Fungal toxins: their biological effects and ecological significance. Presidential Address, 74th Indian Science Congress Association, Bangalore, p. 16. Borut S. Y. and Joffe A. Z. (1966) AspergillusJlauus Link and other fungi associated with stored groundnut kernels in Israel. Israel J. Bot. 15, 112-120. Buchanan R. L. and Lewis D. F. (1984) Regulation of aflatoxin biosynthesis: effect of glucose on the activities of various glycolytic enzymes. Appl. Environ. Microbial. 48, 306310. Buchanan R. L., Ckker L. A. and Stahl H. G. (1985) Effect of 2-deoxyglucose alpha-methyl glucoside, and glucosamine on aflatoxin production by Aspergillus parasiticus. Arch. Microbial. 142, 200-203. Diener V. L. and Davis N. D. (1966) Aflatoxin production by isolates of Aspergillusjluuus. Phyfopulhology 56, 139&l 393. Diener U. L., Cole R. J., Sanders T. H., Payne G. A., Lee L. S. and Kiich M. A. (1987) Epidemiology of aflatoxin formation by Aspergillus Jlavus. Ann. Rev. Phyroputhol. 25, 249-270. ISTA (1966) International rules for seed testing. Proc. Inf. Seed Test. Assoc. 32, I-152. Lemke P. A., Davis N. D. and Creech G. W. (1989) Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species. Appl. Environ. Microbial. 55, 1808-l 8 10. Lee L. S., Lee L. V. Jr, and Russell T. E. (1986) Aflatoxin in Arizona cotton seed: field inoculation of bolls by Aspergillus ~uuus spores in wind driven soil. J. Am. Oil Chem. Sot. 63, 530-532. Lillehoi E. B. (1987) In A&toxin in Maize-A ProceedirwL of, . , The aflatoxin in maize oroblem: the historical uersoective. _ _ the Workshop (Edited by Zuber M. S., Lillehoj E. B. and Renfro B. L.), pp. 13-32. CIMMYT, Mexico. Lillehoj E. B., Knowlek W. F., Homer E. S., Widstrom N. W., Josephson L. M., Franz A. 0. and Catalan0 E. A. (1980) Aflatoxin contamination of preharvested corn: role of Aspergillusjuvus inoculum and insect damage. Cereal Chem. 57, 255-257.
Manabe M. and Tsuruta U. (1978) Geographical distribution of aflatoxin producing fungi inhabiting South East Asia. Jup. Agric. Res. 12, 224-227.
Rao K. S., Madhavan T. V. and Tulpule P. G. (1965) Incidence of toxigenic strains of Aspergillusfiauus affecting groundnut crop in certain coastal districts of India. Ind. J. Med. Res. 53, 11961202. Reddy T. V., Viswanathan L. and Venkitasubramanian T. A. (1970) Thin layer chromatography of aflatoxins. Anulyt. Biochem.
38, 568-57
1.
Sinha K. K. (1990) Incidence of mycotoxins in maize grains in Bihar State, India. Food Addif. Contum. 7, 5541. Stack M. E. and Pohland A. E. (1975) Collaborative study of a method of chemical confirmation of the identity of aflatoxin. J. Assoc. Off. Anulyt. Chem. 58, ll(r113. Waksman S. A. (1952) Soil Microbiology, p. 356. Wiley, New York. Wallbridge A. (1963) Behaviour of different strains of A. j&n~ and related species (Abstr.). UNICEF Conf., Groundnut Toxicity Problems. Tropical Prod. Institute, London, p. I. Wicklow D. T. (1983) Taxonomic features and ecological significance of sclerotia. In Ajluroxin and Aspergillusjluvus in Corn (Edited by Diener U. L., Asquith R. L. and Dickens J. W.), Southern Co-operative Series Bulletin 279, pp. 612. Alabama Agricultural Experiment Station, Auburn, Alabama, U.S.A.
J. stored Prod. Res. Vol. 29, No. 4, pp. 351-355, 1993
$6.00 + 0.00
Copyright0 1993PergamonPressLtd
Printedin GreatBritain.All rightsreserved
IMPACT OF HABITATS ON TOXIGENIC POTENTIAL OF ASPERGILLUS
FLA VU.7
K. S. BILGRAMI and A. K. CHOUDHARY University Department of Botany, Bhagalpur University, Bhagalpur 812007, India (Received far publication
30 March
1993)
Abstract-Toxigenic potentials of Aspergillus@vms strains isolated from different organic substrates, air and soils were evaluated. The frequency of occurrence of A. ~IULUSranged from 76% in maize to 87% (coconut and groundnut) and 92% in makhana (Euryaleferox indica.) Incidence was lowest in green gram (42%). Analysis of variance showed that the percentage incidence of A. ~CIUUSin the aerosphere of maize fields was significantly affected by season x location. In soil samples the frequency of occurrence of A. jZauus was high during the monsoons (76% in non-diara land soils, 69% in diara land soil). Among 1706 isolates of A. $mus obtained from different sources, 826 (48.4%) were found to be toxigenic. The frequency of non-toxigenic strains of A.j?mus was comparatively higher (ratio = 1.07: 1) than the toxigenic strains. Percentage incidence of the toxigenic strains of A. j&nrus was the highest (73.3%; ratio = 0.36: 1) in the soil samples. No attempt was made to differentiate A. jkuus and A. parasiticus and therefore all references to A. fkvus include A. parasiticus. Key words-Aspergillus
jfavus, substrates, soils, aerosphere.
INTRODUCTION
Aspergillus flavus Link contaminates a variety of agricultural commodities with aflatoxins. In previous studies on collections of toxigenic isolates of A. flaws from different natural substrates (Wallbridge, 1963; Austwick and Ayerst, 1963; Borut and Joffe, 1966; Rao et al., 1965; Bilgrami, 1985; Sinha, 1990) the ratio of non-toxigenic and toxigenic strains of A. flaous varied with source and location of isolations. However, there is no direct evidence suggesting a correlation between mycotoxin producing ability and distribution of an isolate in nature. Some indirect evidence suggested for example greater occurrence of cyclopiazonic acid (a secondary metabolite) in the strains of A.JEauus isolated primarily from tropical countries (Manabe and Tsuruta, 1978; Wicklow, 1983). In the present investigation strains of A.JEazw were isolated from diverse habitats viz. cereals, pulses, oilseeds, dry fruits, spices, air (aerosphere of maize fields) and soils. Ratio of the non-toxigenic and toxigenic isolates of A.$uuus in relation to different habitats were evaluated. MATERIALS
AND
METHODS
Seeds of different cereals (maize, wheat, rice, barley), pulses (Cujunus cajun-arhar, Phuseolus mungo-green gram), oilseeds (groundnut, Brussicujunceu-mustard), dry fruits (coconut, Euryule ferox indicu-makhana) and spices (coriander, cardamom and chilli) were obtained from different markets of Bhagalpur and its surrounding areas. For cereals, pulses and oilseeds, each sample comprised at least 1 kg seeds which were collected in polythene bags. The sample size of dry fruits and spices comprised 250 g each. Air and soil samples were collected from different fields of two localities, Locality A was near Sabour Agricultural College, about 8 km east of Bhagalpur University possessed generally loamy, clay-loam soil, comparatively rich in organic matter (0.42-0.8%) and alkaline (pH 7.2-8.2). Locality B, about 12 km west of the University was flood prone diara land, where Khurif crops during harvesting (July-September) remained either fully or partially submerged under flood waters. The soil of this locality was mostly sandy or sand-silt-clay with comparatively lesser organic matter (0.3-0.6%) and was also alkaline (pH 7.2-7.8). Isolation of mycoflora from different substrates was carried out using the blotter test and agar plate methods (ISTA, 1966). All plates were incubated for 7 days. Seeds were examined under the 351
352
K. S. BILGRAMI
and A. K. CHOUDHARY
Table I. Percentage frequency of A. flaws in different cereals, pulses, oilseeds and dry fruits Incidence of A. flow Substrates
Number of samples
Number with A. flmrtrs
% Gccurrence
172 140 60 42
131 58 32 23
76 42 53 55
Cereals
Maize Wheat Rice Barley
Pulses
Arhar Green gram
40 25
27 II
68 44
Oilseeds
Groundnut Mustard
15 72
I3 31
87 51
Dry fruits
Coconut Makhana
30 25
26 22
87 92
Spices
Coriander Cardamom Chilli
30 30 50
21 I9 29
70 63 58
stereo-microscope from the fourth day and the developing mycoflora isolated, purified and maintained on PDA and Czapek-Dox agar medium for further studies. Soil samples were collected from maize fields in both localities from January to September. Each month five random soil samples from each locality were taken at depths of O-3 cm. The soil dilution technique (Waksman, 1952) was used for isolation of soil mycoflora. Plates were incubated at 28 + 2°C for 7 days. After incubation, colonies were counted and the number of fungi per gram of dry weight estimated. The frequency of occurrence of A. flavus was calculated by the following formula: Frequency =
No. of colonies of A.$uvus x loo Total count
Twenty Petri plates containing Czapek-Dox agar were exposed in the fields of both localities from January to September. The duration of exposure ranged from 12-14 h covering from evening to next morning. Exposed plates were incubated (28 +2”C) for 45 days. Isolation and identification of fungi were subsequently carried out. The relative frequency of A. jhvus was noted each fortnight. Statistical analysis (analysis of variance) was performed in order to assess the seasonal incidence of A. JIauus in both the localities. The aflatoxin producing potential of A. frauus isolates was tested in SMKY liquid medium (sucrose-200 g, MgSO, . 7H20--0.5 g, KNO,-3 g, yeast extract-7 g, distilled water-l 1)(Diener and Davis, 1966). Aflatoxin was extracted in chloroform and both qualitative and quantitative analysis carried out. Qualitative detection was by TLC using toluene : isoamylalcohol : methanol (90: 32: 2 v/v/v) as solvent system (Reddy er al., 1970). Quantitative assays of aflatoxin were Table 2. Incidence of A. jidw.r in the aerosphere of maize fields at Bhagalpur (2-way table) % incidence of Aspergillw J?avus in different seasons Season
Winter (Jan-Mar) Summer (Apr-Jun) Monsoon (Jul-Sep) Mean
Locality A(L,)
Locality B(L,)
Mean
23.2 (28.5) 35.5 (32.3) 66.1 (54.6) 41.6 (39.8)
23.0 (28.1) 52.7 (46.5) 59.8 (50.8) 45.2 (41.8)
23. I (28.3) 44.1 (41.4) 63.0 (52.7) 43.4 (40.8)
Figures in parentheses indicate transformed values. Since the original values were in percentage, these were therefore, transformed into degrees and then analysis of variance was done. (Angle =arc sin Js where, L, = Non-diara land and L, = Diara land). 5% CD for season = 5.322. 5% CD for season x location = 7.526. (Note : Analysis of variance was performed in order to evaluate the effect of locality and interaction effect of season x location for the incidence of A. flows in the aerosphere of maize fields.)
Impact of habitats on toxigenic potential of A. Jlauus
353
Table 3. % Relative density of A. fIouw in the soils of Bhagalpur Relative density of A. @wus (%)
Average number of fungus/g dry wt Diara land of soils soil (Locality B) (Fungi x 10s)
Non-diara land soil (Locality A)
Seasons Winter (Jan-Mar) Summer (Apr-Jon) Monsoon (Jul-Sep)
38
54
5.2
33
21
4.8
76
69
8.6
determined spectrophotometrically (AOAC, 1984). Chemical confirmation was by trifluoroacetic acid (Stack and Pohland, 1975). In the present investigation, no attempt has been made to differentiate A. j&.ruus from A. parasiticus, so all references to A. frauus include A. parasiticus.
RESULTS
Aspergillusjlavus, A. niger, A. ochraceus, Aspergillus spp., Penicillium citrinum, other Penicillium spp., Fusarium moniliforme, F. equiseti and other Fusarium spp. were the most frequent fungi in different substrates. Other common fungi included Rhizopus stolonifer, Mucor spp., Alternaria spp., Cur&aria spp., Drechslera spp., Chaetomium spp., Lasiodiplodia theobromae and sterile mycelia. Infection rates varied from 24% (green gram) to 62.7% (maize). The frequency distribution of fungal species isolated from the various sources varied substantially. Aspergillusflauus was the dominant fungus with its frequency ranging from about 76% in maize to 87% in coconut and groundnut and 92% in makhana (Table 1). Its incidence was lowest in green gram (42%). It is clear from the analysis of variance (Table 2) that the percentage incidence of A. flaws in the aerosphere of maize fields was significantly affected by season x location. However, location alone did not have any significant effect. Maximum incidence of A.Jlavus was recorded during the monsoon season (63%) followed by summers (45%) and winters (24%). These differences were statistically significant. Crops grown in locality B (flood prone diara land) had much higher incidence of A. frauus during summers (53%) than in locality A (dry area, 36%). In the soil samples, the frequency of isolation of A. flaws was also high during the monsoons (76% in locality A, 69% in locality B) (Table 3). The lowest incidence (21%) of A. flavus was observed during the summers in locality B. Average number of fungi per gram dry weight of soils varied from 4.8 x lo8 to 8.6 x 10’. Of 1706 isolates of A. flaws obtained from different sources screened for aflatoxin production in SMKY liquid medium (Table 4), 826 (48%) isolates were found to be positive. Overall, the Table 4. Aflatoxin oroducine ootentialities of A. Aavus obtained from different habitats
A
B
C
Ratio of nontoxigenic and toxigenic isolates
240 90 130 I80 85 96 II0 185 120 60 60 60 60 110 120 I706
149 38 72 70 36 35 63 77 61 26 23 I9 24 45 28 826
62 42.2 55.4 39 42.4 36.5 57.3 41.6 50.8 43.3 38.3 31.7 40 40.9 73.33 48.4
0.61: I 1.37: I 0.81: I 1.57: I 1.36: I 1.74: I 0.75: I 1.4O:l 0.96: I 1.31:l 1.60: I 2.16: I 1.5:l l.4:1 0.36: I 1.07:l
Number of isolates Source of isolates 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Maize Wheat Rice Barley Arhar Green gram Groundnut Mustard Coconut Makhana 11.Coriander 12. Cardamom 13. Chilti 14. Aerosphere 15. Soils Total :
No. of isolates producing allatoxins B,
B, + B,
42 7 I7 15 II I2 18 34 19 4 5 5 II 14 28 242 (29.3%)
67 la 31 34 I4 I9 33 28 I7 I6
1 I2 6 8 I7 321 (39%)
B,+B,+G, 35 13 20 21 9 4 9 12 23 6 17 2 7 23 32 233 (28.2%)
B,+B,+G,+G, 5 4 2 3 3 2
II (3.&
Amount of aflatoxin (ppm) Min. I trace 2 2 trace trace 3 trace 7 5 trace 3 trace 2 a trace
Where A = Number of isolates screened, B = Toxigenic isolates, C = % toxigenic isolates, Min. = Minimum, Max. = Maximum.
Max. 21 15 18 I8 I3 18 I8 20 23 la I? I2 7 21 26 26
354
K. S. BILGRAMI and A. K. CHOUDHARY
incidence of non-toxigenic strains was higher and the ratio of non-toxigenic and toxigenic strains of A. ftavus was 1.07: 1. While considering different sources of isolation, percentage incidence of the toxigenic strains of A.Juuus was the highest (73.3%) in the soil samples, where the ratio of non-toxigenic and toxigenic isolates was 0.36: 1. The amount of aflatoxin produced by the isolates obtained from the soil was also a maximum, averaging 26 ppm in the liquid medium used. The incidence of the toxigenic isolates of A. flauus varied considerably from the different substrates. Toxigenic isolates of A.$avus ranged in cereals from 40 to 62%, in pulses 36.5 to 42.4%, in oilseeds 41.6 to 57.3%, in dry fruits 43.3 to 51%, in spices 32 to 40%, while in air it was 41% (Table 4). Carbohydrate rich materials had comparatively higher incidences of toxigenic isolates. Among the cereals, the incidence of the toxigenic isolates was highest (62%) in maize. Aflatoxin B, production of A. flavus strains isolated from different substrates was highest in the strains obtained from coconut (23 ppm) followed by maize (21 ppm). It was low by the isolates obtained from chilli (7 ppm) and cardamom (12 ppm). Toxigenic isolates of A. fEavus which were distributed in wide range of habitats, elaborated varying fractions of aflatoxin in liquid media. Aflatoxin B, was produced by all the toxigenic isolates of A.Jlavus (826), whereas, 242 (29.3%) isolates could produce aflatoxin B, only. 321 (39%) toxigenic isolates were capable of elaborating both aflatoxin B, and B,, while 233 (28.2%) isolates produced B,, B2 and G,. All the four components of aflatoxins (viz. B,, Bz, G, and G,) were produced by 30 isolates only (3.4%). None of the isolates, however, were able to elaborate aflatoxin B,, G, or Gr in absence of B,. The range of aflatoxin production in different isolates of A.flavus varied from trace to 26 ppm.
DISCUSSION A high incidence of A. fravus in the aerosphere of maize crops at and around Bhagalpur was observed during the monsoons followed by summers. Correlation between air-borne inoculum of A. fravus with aflatoxin contamination in fields has been reported earlier (Lillehoj et al., 1980; Lee et al., 1986). Crops grown in locality B had a much higher incidence of A. flavus during summers (53%) than in locality A (36%), perhaps due to extreme scarcity of water (Diener et al., 1987). Large frequency of occurrence of A.&vus were observed in the soils during the monsoons. This is in conformity with the results of Angle (1987) who reported a high incidence of A. jSzvus in the soil samples. In soils, plant residues of susceptible crops (viz. maize, peanuts) have been reported as congenial focal points for development of A. fiavus (Lillehoj, 1987). Because it is known that A. JEavus produces only B aflatoxins, it can be deduced that 233+30 = 263 (ca 30%) of the toxigenic isolates were A. parasiticus. As A. parasiticus is always toxigenic, 26311706, i.e. 15.4%, were A. parasiticus. While taking different habitats into account, it was observed that the frequency of non-toxigenic strains of A. JEavus was comparatively higher (ratio 1.07: 1) than the toxigenic ones. Considerable variations were observed in the distribution of non-toxigenic and toxigenic isolates of A. j&us on different organic substrates/air/soils. Bilgrami (1987) has suggested that the non-toxigenic forms are the wild type while the toxigenic ones are of mutant types. Mutants by and large have lower selective advantage due to increased genetic load in them, therefore, their number is lesser as compared to the non-toxigenic ones. Lemke et al. (1989) on the basis of single spore cultures of A. flavus on several succeeding generations have also confirmed it. Among different organic substrates, carbohydrate rich seeds were quite favourable abode for the selection of the toxigenic isolates of A. Jhzvus. Abdollahi and Buchanan (198 1) have suggested that maximum toxin yields are dependent on high concentrations of specific carbohydrates. It has been argued that aflatoxin biosynthesis may be regulated by a carbon-catabolic induction process, in which the built up of the key intermediates or alterations of energy status of the cell leads to the induction of aflatoxin synthesis (Buchanan and Lewis, 1984; Buchanan et al., 1985). Fatty acids and carbohydrate contents or some toxin stabilizing factor in coconut might be responsible for selection of highly toxigenic strains of A. J&zvus.
Impact of habitats on toxigenic potential of A. Jlavus
355
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