Mycobiota and toxigenic Aspergillus flavus associated with developing cardamom and pepper

Mycol. Res. 97 (11): 1403-1406 (1993)

1403

Printed in Great Britain

Mycobiota and toxigenic Aspergillus {lavus associated with developing cardamom and pepper

AMEET A BANERJEE, RANJI P. MA THEWS, H. S. PRAKASH AND H. S. SHE TTY Department of Studies in Applied Botany, University of Mysore, Mysore 570006, India

Cardamom and pepper were infected by Fusarium moniliforme, F. solani and Aspergillus f/avus at different stages of development, The immature fruits were highly susceptible to colonization by Fusarium which resulted in rotting of the berries and capsules at early stages in their development, Subsequently, they served as a good substrate for colonization by species of Aspergillus, Penicillium and Fusarium, Other fungi which occurred in small numbers were Trichoderma, Pestalotia, Rhizopus, Cladosporium as well as some actinomycetes, Aflatoxin production in vitro by A. f/avus, isolated at various stages of development, was assessed using a MAb-based ELISA. All Aspergillus f/avus isolates tested produced aflatoxin B1 in amounts ranging from 65-3000 ng ml- 1 ,

Pepper (Piper nigrum L.) and Cardamom (Elettaria cardamom M,) are the most important spices in India, Although contamination of spices with A. flavus has been known (Flannigan & HUi, 1976), no attempt has been made so far to study the frequency of its occurrence in developing fruits of cardamom and pepper, Such information is essential in maintaining the quality of spices, and for implementing appropriate control measures during development or processing, Recently, there have been reports on the occurrence of aflatoxin in Indian spices particularly, in pepper and cardamom (Seenappa & Kempton, 1980; Neumeyer & Forstmeier, 1981; Madhyasta & Bhat, 1984, 1985), Seenappa & Kempton (1980) reported that naturally contaminated black pepper from Kerala, India contained 15-25 (.Ig kg -1 of aflatoxin B1 , Suzuki, Danius & Kilbuck (1973) found 1'8-3'7 ppb of aflatoxin G 1 and 1'1 ppb of aflatoxin G 2 in ground black pepper but reported that cardamom contained no aflatoxins, However, details of the analytical methods used for the detection of aflatoxin B1 were limited,

Existing methods for aflatoxin detection and quantification in spices are not very sensitive and are somewhat complex. Thin layer 2-dimensional chromatography can detect 1'3-2'5 (.Ig aflatoxin in spices (Scott & Kennedy, 1973), whereas an enzyme linked immunosorbent assay (ELISA) based on monoclonal antibodies (MAbs) can detect 5 ng g-1 of the toxins in samples (Ramakrishna et aI., 1990), In the present study an attempt was made to identify and enumerate the mycobiota associated with developing pepper and cardamom and to assess the ability of isolates of A. flavus from these spices to produce aflatoxin B1 using a MAb-based ELISA.

MATERIALS AND METHODS

Collection of plant material Three samples of cardamom at different stages of maturity were collected from three different sites at Wynad, Kerala at approximately one month intervals from 6 September to 15

Table 1. Percentage incidence of mycobiota on cardamom flowers and capsules at various stages of development and processing

Mature fruit

Immature fruit

Aspergillus flavus A. niger A. ochraceus Cladosporium cladosporioides Fusarium moniliforme F. solani Penicillium spp, Trichoderma viride Actinomycetes Values are mean ±

S,E,

Harvested and processed capsule

Dry capsule

Inflorescence

Fruitwall

Seed

Fruitwall

Seed

Fruitwall

Seed

Fruitwall

24'O±4'7 16'6±2'7 0 16'6±2'7

16'6±2'7 7'3±3'O 0 23'3±S'4

22'6±3'7 16'6±8'S 0 1J03±2'7

13'3±2'7 20'6±4'2 0 0

16'O±O'8 lS'O±O'S 0 0

16'O±2'5 13'3±2'7 0 0

16'6±S'4 21'O± 1'7 0 0

73'3 ± 2'7 17'3 ± 4'3 36'6±2'7 7'3±3'O 10'6±O'S 10'6±4'3 9'6±1'2 8'3±O'7

3'3±2'7 31'6±7'6 1J03±2'7 0 0

43'3± IS'l lO'6±O'S 0 9'3±O'S 0

23'0±6'3 lO'6±l'O 0 0 0

l'6±O'9 13'3 ±2'7 S'O±2'9 0 0

16'6±S'8 S'6±3'S 4'3±3'S 0 0

lO'O±4'7 19'3±O'S 11'6±1'3 7'3±3'O 0

30'1±6'9 16'6±1'9 13'3 ± 1'9 6'3±3'3 0

26'6±2'721'O±6'S 13"3 ± 2'7 10'3 ± 1'3 IS'6±3'S 11'6±2'1 10'6±O'S 4'6±3'S 13'3±2'710'3±4'2

of three samples.

Seed

Aspergillus flavus on cardamom and pepper

1404

Table 2. Percentage incidence of mycobiota on pepper flowers and berries at various stages of development and processing Developmental stage/fungal incidence (%) Age of berry (in months) Inflorescence

Aspergillus flavus A. niger A. ochraceus A. candidus A. verscicolor Fusarium moniliforme F. so/ani Penicillium spp. Pesta/otia sp. Rhizopus stolonifer Trichoderma viride Aetinomycetes Values are mean ±

S.E.

a lO'O±4'7

a a a

One

Two

Three

Four

28'6±3'S

21'3 ± 4'1 3-3±O'7 1I-O±O'4

21'6±2'8

22'O±4'l

21'O±l'2 1O'0±0'8 6'0±O'8 0 4'3±O'9 0 0

21'S±0'8 1O'3±4'2 0 0 8'O±1'2 0 0

a a a a

26'6±9'8 Il'6±3'6

24'0±O'9 I4'6±O'7

20'O±4'7

0 S'6±O'7

a a

23'3±2'7 0

a

a a

a a

a

6'O±O'3

a a

a a a a

22'6±O'8 8'0 ± 3-3 0

a

S'0±l'6 0 0

After harvest and processing 3I-3±3'8 Il'O±O'4 2l'O±4'O I4'O±2'4 4-3 ± 1'9 9'6±O'7 4'3±O'7 1O'6±3-5 0 0 0 8'6± 1"4

of three samples.

December 1990, Pepper samples were also collected from three location in Wynad district at different times from July to November. In both cases the samples were taken at stages ranging from flowering to mature fruits, They were collected in sterile polythene bags, brought to the laboratory and stored at 5 0c.

Mycobiota analysis Cardamom, Twenty flowers were removed from the inflorescence from each sample, surface-sterilized for 2 min in 1% sodium hypochlorite solution and plated on potato dextrose agar (PDA) and Czapek-Dox agar (CDA), Young, mature and dried capsules, fruit wall and seeds were separated with sterile forceps and plated on the same media, Pepper, Inflorescences, immature and mature berries were surface-sterilized and plated on PDA (10 per plate), Samples of cardamom and pepper were also plated on CDA after harvest and processing to facilitate detection of A. flavus, The plates were incubated at 25 ± 2° and observations were made after 5 d,

Culturing of A. flavus and preparation of inoculum Isolates of Aspergillus flavus (15 from pepper and 20 from cardamom) were maintained on PDA slants and grown in a modified Czapek-Dox liquid medium, Inoculum was prepared by flooding 10-d-old cultures with sterile distilled water containing 5 % Tween 20 (vIv) and gently agitating the cultures to detach the spores, The spore concentration was adjusted to 2 x 10 4 conidia ml- 1 using a haemocytometer and 0'5 ml of the suspension was added to 100 ml of the medium in 250 ml conical flasks, three replicates for each isolate. The flasks were incubated at 25°C for 7 d and then the culture broth was filtered through Whatman No, 41 filter paper, Aflatoxin B1 was extracted from culture filtrates by mixing with acetonitrile/potassium chloride (0'5 %)/sulphuric acid

(6%), (80:10:1, v/v) solution in a Waring blender for 2 min, The extract (100 1J1) was diluted in a ratio of 1: 40 (vIv) with buffer (9% acetonitrile in Tris HCl Buffer, pH 7'4) and used directly in ELISA.

Quantification of aflatoxin B, Aflatoxin B1 in the culture filtrate was quantified using a MAb-based indirect ELISA following the method of Ramakrishna et al. (1990), The monoclonal antibodies to aflatoxin B1 developed by Candlish et al. (1985) at the University of Strathclyde, Glasgow (marketed by RhonePoulenc, Diagnostics), were kindly provided by Dr John Lacey Rothamsted Experimental Station, Harpenden, Herts, U.K, RESULTS

Mycobiota Cardamom, The incidence of various fungi at different stages of development of cardamom fruits are shown in Table 1. A small number of fungi which occurred at all stages from inflorescence to processed capsule were predominant: Aspergillus flavus Link ex Gray, A. niger van Tieghem, Fusarium moniliforme Sheld, and F. solani (Mart.) SacCo A. flavus was isolated from 24 % of inflorescences and at similar levels at all other stages of development except in the fruit wall of processed capsules where it reached 73 %. The pattern for A. niger was generally similar though usually at a lower incidence throughout. Fusarium moniliforme was only isolated from 3 % of inflorescences but it was very common in the walls of immature fruit and at later stages of development unlike F. solani which was most commonly isolated from the inflorescence (32 %), Infection of developing fruits by F. moniliforme and F. solani caused rotting of capsules and berries. Other fungi and microorganisms were found only intermittently and rarely above 15 %. Thus A. ochraceus Wilhelm and actinomycetes were only found in parts of the processed capsule (10-15 % in both cases), In contrast, Cladosporium

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A. Banerjee and others

cladosporioides was present on flowers (17%), immature (23%) and processed fruits, but was absent from mature fruits and dry capsules, and various Penicillium spp. were isolated from inflorescences (13 %) and from the mature fruit stage onwards but not from immature fruit. Trichoderma viride was present mainly at later stages of development although it was also isolated from the walls of immature fruit (95 %). Pepper. A different range of fungi was isolated from pepper than from cardamom but the principal components of the mycobiota were the same in both cases (Table 2). A. flavus was the commonest of five Aspergillus spp. and was isolated from all stages of pepper development except inflorescences. It was also the fungus most frequently isolated from processed berries and Aspergillus spp. together, were the dominant group on processed berries. As with cardamom, A. niger was present on inflorescences. Both F. moniliforme and F. so/ani were isolated from all developmental stages, the former being generally the commoner of the two, although levels were lower on processed berries than at all other stages. Other fungi, including Penicillium spp., Pesta/otia spp. and Rhizopus st%nifer, were present at various stages but were absent from processed berries.

Production of aflatoxin 8 1 The isolates of A. flavus isolated from different stages of developing cardamom produced aflatoxin B1 ranging from 100 to 3000 ng ml- 1 medium. Seven produced more than 100 ng aflatoxin ml-\ 12 produced more than 1000 ng ml- 1, and one from processed cardamom produced more than 100 ng ml- 1 of medium. All 15 isolates of A. flavus from developing pepper also produced aflatoxin B1 but they varied considerably in amounts produced. Two produced less than 100 ng ml-\ 11 more than 100 ng ml- 1 , and one more than 500 ng ml- 1 of medium. One of the isolates from 2-month-old berries produced 3000 ng ml- 1 .

DISCUSSION Cardamom sampled at different growth stages was heavily colonized by fungi, mainly species of Aspergillus including A. flavus, A. niger and A. ochraceus, Penicillium spp. and Fusarium spp. Aspergillus and Penicillium spp. not only contaminated cardamom after processing, but also infected the immature crop prior to harvest. The presence of A. flavus on the fruit wall and seeds in large numbers suggests that the inoculum of A. flavus could have come from the soil or from the air. At maturity when the capsules of cardamom had dried, A. flavus was the most dominant fungus both on the fruit wall and on seeds. The analysis of mycobiota on developing pepper again showed the predominance of Fusarium spp. and Aspergillus spp. indicating a remarkable similarity in the spectrum of moulds found on cardamom and pepper at different de-

velopmental stages. Dried pepper, after processing, showed a preponderance of A. flavus following by A. niger, A. candidus, A. ochraceus, Penicillium spp. and actinomycetes, confirming and extending the observations of Flarmigan & Hui (1976) who observed the presence of strains of A. flavus capable of producing aflatoxin B1 in ground spices. F. moniliforme and F. solani occurred in low numbers. A. flavus isolates from pepper clearly possess the potential to colonize the developing pepper and produce aflatoxin B1 . Some investigation on the mycotoxins produced by the Fusarium spp. may also be merited. Most of these fungi can apparently invade the pepper fruit before harvest despite pulpy flesh and heavy skin. Christensen et aI. (1967) had suggested that the nature of the pepper fruit might not allow the invasion of storage fungi before harvest. From the present study, it is clear that the aflatoxin producing aspergilli can colonize the fruits during their development. In addition, faulty post-harvest practices like incorrect harvesting, improper drying and storage and poor quality processing may lead to further contamination with aflatoxin. The ability of Aspergillus and PenicilIium spp. to infect the immature crop and contaminate the processed crop, together with the capability of A. flavus isolates to produce aflatoxin B1 in amounts ranging from 65-3000 ng ml- 1, is a cause for concern in India, the largest producer and exporter of cardamom and pepper. Aflatoxin B1 could be formed at any stage in seed development and accumulate in the processed spices. Thus, there is a need for a simple, rapid and sensitive method to detect aflatoxin. B1 and other toxins in spices. So far there are no reports on the accumulation of aflatoxin B1 in developing fruits. Further work along these lines will be done using the MAb based ELISA to detect aflatoxin B1, which in the present study was very efficient owing to its simplicity in use, specificity and sensitivity.

The senior author (A. B.) wishes to acknowledge the financial support of Council of Scientific and Industrial Research, New Delhi.

REFERENCES Candlish, A. A. G., Stimson. W. H. 8< Smith, J. E. (1985). A monoclonal antibody to aflatoxin B,: detection of the mycotoxin by enzyme immunoassay. Letters in Applied Microbiology 1. 57-61. Christensen. C. M .• Fanse. H. A.. Nelson, G. H., Bates. F. 8< Mirocha. C. j. (1967). Microflora of black and red pepper. Applied Microbiology 15, 622-629. Flanningan. B. 8< Hui. S. C. (1976). The occurrence of Aflatoxin producing strains of Aspergillus f/avus in the mould floras of ground spices. Journal of Applied Bacteriology 41. 411-418. Madhyasta. M. S. 8< Bhat. R. V. (1984). Aspergillus parasiticus growth and aflatoxin production on black and white pepper and the inhibitory action of their chemical constituents. Applied and Environmental Microbiology 48. 376-379. Madhyasta. M. S. 8< Bhat. R. V. (1985). Aflatoxin like fluorescent substances in spices. Journal of Food Safety 7. 101-106. Neumeyer. L. 8< Forstmeier. G. (1981). Distribution of microorganisms in and on spices (pepper). Fleisch Wirtschaft 61, 630-632.

Aspergillus flavus on cardamom and pepper Ramakrishna, N., Lacey, )., Candlish, A. A. G., Smith, J. E. & Goodbrand,). A. (1990). Monoclonal antibody based enzyme linked immunosorbent assay of aflatoxin B" T-2 toxin and ochratoxin A in Barley. Journal of the Association of Official Analytical Chemists 73, 71-76. Scott, P. M. & Kennedy, B. P. C (1973). Analysis and survey of ground black, white and capsicum pepper for aflatoxin. Journal of the Association of Official Analytical Chemists 50, 1452-1457.

1406 Seenappa, M. & Kempton, A. G. (1980). Application of a minicolumn detection method for screening spices for aflatoxin. Journal of Environmental Science and Health. Part B. Pesticidees, Food Contaminants and Agricultural Wastes 15,219-231. Suzuki, S. L, Dainius, B. & Kilbuck, ). H. (1973). A modified method for aflatoxin determination in spices. Journal of Food Science 38, 949-950.

(Accepted 29 April 1993)

PHYTOPHTHORA Edited by J. A. LUCAS, R. C. SHATTOCK, D. S. SHAW & LOUISE COOK The seventeenth symposium of the British Mycological Society was held jointly with the British Society of Plant Pathology and the Society of Irish Plant Pathologists. The subject of the symposium was Phytophthora, the organism responsible for many plant diseases, most notably potato blight. This book presents the results of the meeting, in a wide-ranging volume incorporating chapters discussing the history of potato blight, host-pathogen reactions, systematics and intraspecific variation, molecular and genetic studies and the development of effect control methods. Arguably the most studied of all plant pathogens. Phytophthora remains a problem in modem agriculture.

Contents 1. The Rev. Miles Berkeley and the blight of the potato; 2. Potato blight in Europe in 1845: the scientific controversy; 3. Phytophthora infestans: the Mexican connection; 4. Host-pathogen interactions; 5. Ultrastructural and immunological studies of zoospores of Phytophthora; 6. Surfacerelated host-pathogen interactions in Phytophthora; 7. Molecular aspects of host-pathogen interactions in Phytophthora; 8. Current questions in Phytophthora systematics; 9. Relationships between nonpapillate soilborne species of Phytophthora root rot of raspberry; 10. Variation in the species of the Phytophthora megasperma complex; 11. Molecular approaches in Phytophthora taxonomy using polymorphisms in mitochondrial and nuclear DNA; 12. Relationships between Phytophthora species: evidence fom isozyme analysis; 13. Molecular mapping in Phytophthora infestans; 14. Variation in ploidy in Phytophthora infestans; 15. Inheritance of virulence and other phenotypic traits in Phytophthora infestans; 16. Isozymes in Phytophthora infestans; 17. Parasexual genetics in Phytophthora; 18. Restriction fragment length polymorphisms in Phytophthora infestans; 19. Transformation in Phytophthora parasitica; 20. Towards gene-transfer systems and understanding gene structure in Phytophthora infestans; 21. Forecasting and control strategies for potato late blight; 22. Current problems in chemical control of late blight: the Northern Ireland experience; 23. Phenylamides and Phytophthora; 24. Synergism among fungicides for control of Phytophthora; 25. Development and evaluation of blight resistant potato cultivars; 26. Microbial suppression of Phytophthora cinnamomi; 27. Chemical and biological control of Phytophthora species in woody plants; 28. Strategies for the integrated control of soilborne Phytophthora species; Index.

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