Bot. Bull. Acad. Sin. (1995) 36: 229-234

Tseng et al. — Mycoflora and mycotoxins in bean

Mycoflora and mycotoxins in dry bean (Phaseolus vulgaris) produced in Taiwan and in Ontario, Canada

T. C. Tseng1,4, J. C. Tu2, and S. S. Tzean3

1Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China

2Agriculture and Agri-Food Canada Research station, Harrow, Ontario, NOR IGO, Canada

3Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, Republic of China

(Received May 17, 1995; Accepted July 19, 1995)

Abstract. We undertook a comparative investigation of mycoflora and mycotoxins in dry bean (Phaseolus vulgaris) produced in Taiwan and Ontario. Seedborne fungi were isolated and characterized. A profile of bean mycoflora was developed for each region having distinct climatic conditions. The frequency of various isolated fungi was also determined. The average percentages of seedborne fungi in the bean lots from Ontario and Taiwan were 54.8 and 58.5%, respectively. The fungi most-frequently isolated from the diseased Ontario beans were Alternaria (61.1%), Fusarium (18.0%), Rhizoctonia (6.1%), Penicillium (5.2%), Rhizopus (3.2%), Sclerotinia (3.0%), Gliocladium (2.2%), and Mucor (1.7%). The fungi most-frequently isolated from the diseased Taiwan beans were Aspergillus (48.5%), Penicillium (27.6%), Eurotium (6.7%), Rhizopus (5.3%) and Curvularia (2.4%). Based on these profiles, Fusarium and Aspergillus were identified as the most-probable mycotoxin-producing fungi in the bean lots from Ontario and Taiwan, respectively. Subsequent analyses were made of the fungal-infected and non-infected beans from each region. The infected beans from Ontario contained the fusarium toxins diacetoxyscripenol, deoxynivalenol, T-2 toxin and fumonisin B1, but no aflatoxins. The infected beans from Taiwan contained aflatoxins B1, B2, G1, and G2, but no fusarium toxins. In the non-infected bean samples from both regions, however, neither fusarium toxins nor aflatoxins were detected. The monitoring of mycotoxins in imported beans can be simplified by developing a profile of the mycoflora from each exporting country. The profile can help to narrow the range of mycotoxins to be detected. A similar approach can be taken for other field crops.

Keywords: Mycoflora; Mycotoxins; Phaseolus vulgaris; Seedborne fungi.


Mycotoxins are secondary metabolites of fungi. They are toxic and/or carcinogenic. Their presence in foods and food products is a serious health hazard to consumers (Betina, 1989). Seedborne fungi are the principal producers of mycotoxins. Contaminated agricultural products—cereals and oil seeds in particular—are the main sources of mycotoxins in the animal and human food chains. Clearly, mycotoxins are the result of fungal growth on crops in the field and in storage (Scott et al., 1985).

Taiwan has evolved from an agricultural society into a highly industrialized society. Its economy is dominated by manufacturing and trading, and it has one of the highest population densities (approximately 580 km-2) in the world. Land is costly and has been lost rapidly to industrial use and infrastructural development. Production of low-price field crops has decreased sharply. Large quantities of field-crop products are imported to meet domestic needs. The presence of mycotoxins in imported agricultural products is a pressing concern, and careful monitoring is needed to safeguard consumers. It is widely acknowledged that Aspergillus, Penicillium, and Fusarium are the most impor

tant mycotoxin-producing fungi (Betina, 1989). The species in these genera range from primary to opportunistic pathogens to saprophytes. Many species are notorious for their mycotoxin production (Betina, 1989; Chelkowski, 1989). Mycotoxins produced by these fungi have been well investigated in several cereals and oil seeds, such as wheat, corn, and peanut. Unfortunately, little attention has been paid to various dry beans in the genera of Phaseolus, Vicia, and Vigna, all of which are destined for human consumption.

The presence of mycotoxins in bean has been suspected. In Ontario, for example, 18% of the bean crop was reportedly infected by Fusaria in a wet harvest season (Tu, 1989). A few infected bean samples with distinct discoloration were analyzed by a toxicology laboratory. An average of 16 µg g-1 of T-2 toxin was found ( J. C. Tu, unpublished data). Recently, many bean samples with fusarium infection were analyzed and found to contain DAS (diacetoxyscripenol), DON (deoxynivalenol), T-2 (T-2 toxin), and FB1 (fumonisin B1) (Tseng et al., 1995).

Detection of mycotoxin is costly and time consuming, and the methods and procedures of detection are diverse—some mycotoxins require highly-specific methods. Moreover, each genus of Aspergillus, Penicillium, and Fusarium produces a host of specific mycotoxins. In short, the presence of a mycotoxin in a product may not be detected if

4Corresponding author.

Botanical Bulletin of Academia Sinica, Vol. 36, 1995

Aflatoxin—We closely followed the extraction and clean up procedures described by Lu and Ling (1974).

Toxin Identification

Fusarium mycotoxin—The identification of DAS (diacetoxyscripenol), DON (deoxynivalenol), T-2 (T-2 toxin) and FB1 (fumonism B1) was performed by TLC (thin-layer chromatography). With the exception of FB1, which was identified by the TLC method of Gelderblom et al. (1988), the identification methodology followed Romer (1986), with a few minor modifications. The concentrated sample extracts and standards were spotted on TLC plates (aluminum silica gel 60, Art. 5553, Merck, Germany) and developed in toluene-ethyl acetate-88% formic acid (6+2+1) until the solvent front was 1 cm from the top of the plate. The plates were sprayed with 20% AlCl3 in MeOH and air-dried. The dried plates were heated at 110°C for 10 min. The toxins were identified as fluorescent spots at the same height (Rf value) as the standard spots under an ultraviolet (365 nm) lamp. Quantification of DAS, DON, and T-2 was accomplished by GC (gas chromatography), and that of FB1 by HPLC (high performance liquid chromatography).

For GC, we used a Hitachi model 163 gas chromatograph equipped with a flame ionization detector, and closely followed the GC procedures used by Scott and Kanhere (1986). The sensitivity of the GC method for quantification of DAS, DON, and T-2 was less than 40 ng g-1.

For HPLC, a modification of the procedure of Shephard et al. (1990) was used. Briefly, OPA (o-phthalaldehyde) reagent was prepared by dissolving OPA (40 mg) in methanol (1 ml) and adding 0.1 M sodium borate (5 ml) and 2-mercaptoethanol (50 µl). Derivatives of fumonisins were prepared immediately before injection by adding OPA reagent (175 µl) to the sample solution (25 µl) and 0.1 M sodium borate (25 µl). The derived samples were analyzed by a reverse-phase, isocratic HPLC system consisting of a Water Associates (Milford, Massachusetts, USA) model 6000A solvent delivery system and U6K injector. The analytical column (Lichrosorb RP-18 [10 µm], 250 × 4 mm, Art. 50334) and a pre-column filter (Lichrosorb RP-18 [7 µm])) were purchased from Merck, Germany. The detector was a model FS 970 fluorometer (Schoeffel Instrument Corp., Westwood, New Jersey, USA). Excitation and emission wavelengths were 335 nm and 440 nm, respectively. Quantification by peak height measurement was performed with a Model TR-250 Toricorder (Tokyo Rikokikai Co. Ltd., Japan). The eluate was methanol : 0.1 M sodium dihydrogen phosphate (80/20; v/v) adjusted to pH 3.3 with phosphoric acid. The flow rate was 1 ml min-1. The detection limit of the HPLC system for FB1 was 40 ng g-1.

Aflatoxin—The qualitative analysis of aflatoxins B1, B2, G1, and G2 was performed by TLC in accordance with the method of Lu and Ling (1974). The results were confirmed by HPLC with a normal phase, isocratic system as mentioned above. The analytical column (Lichrocart 250-4 Lichrospher Si60 [5 µm] Art. 1. 50830, Merck, Germany)

the detection method is not specific to that toxin. Since mycotoxins are the result of fungal infection and growth, knowing the mycoflora in the infected bean sample should provide a clue to the most-probable toxins.

In this paper, we report the relationship between specific toxins and specific fungal genera in fungal-infected beans produced in two regions where climate and cultural practice differ strikingly. Predetermination of mycoflora in a given crop produced in a given region can accurately predict the presence of certain mycotoxins, and consequently reduce the number of detection methods needed for a product.

Materials and Methods

Isolation and Characterization of Seedborne Fungi

A total of 40 bean samples were collected, 20 from each of Ontario and Taiwan. For fungal assaying, four batches of 50 seeds were surface sterilized for 2 min in 0.5% sodium hypochlorite and then rinsed twice in sterile water. The seeds were cut in half and placed on PDA (potato dextrose agar) plates containing 40 µg ml-1 of tetracycline to inhibit bacteria, 5 half-seeds per plate. Four days later, the fungi that grew out of the seeds were isolated and identified. Aspergillus, Penicillium, and Fusarium were identified based on their morphology on differential media according the classification schemes of Booth (1971), Nelson et al. (1983), Chelkowski (1989), Piff (1979), Raper and Fennell (1965), and Tzean et al. (1990). All the other fungal isolates were cultured on PDA and identified morphologically, referring to Ames (1961), Domsch et al. (1980), Ellis (1971), Hesseltine and Fennell (1955), Liou and Chien (1990), Matsushima (1975), and O'Donnell (1979).

Sample Preparation

Fungi-infected beans from bean lots from Ontario and Taiwan were identified visually. The infected beans and the healthy beans of each category were separated. Each sample was ground to a fine powder in a mill (Ye-Shin Iron Factory, Taoyuan, Taiwan) for 3 min, and 25 g of each sample was extracted for analysis of fusarium mycotoxins and aflatoxins. All experiments were performed three times. Data presented are the average of values.

Sample Extraction and Purification

Fusarium mycotoxin—The sample was extracted in an Ultra Turrax T-25 blender (Janke & Kunkel Gmbh & Co. Germany) with 100 ml of CH3CN-water (84+16) at 9,500 r.p.m. for 3 min and filtered through Whatman No. 4 filter paper. We followed the purification procedures prescribed by Romer (1986) for the determination of trichothecenes Type A and B. All solvents were first-class reagent-grade purchased from Wako Chemical Industries Ltd., Japan and ALPS Chemical Co., Taiwan. The charcoal/alumina cleanup columns (MycoSep #225) were purchased from Romer Labs, Inc., (Union, Missouri, USA).

Tseng et al. — Mycoflora and mycotoxins in bean

were purchased from Merck, Germany. The detector was a model F-1050 fluorometer (Hitachi, Japan). Excitation and emission wavelengths were 360 and 425 nm, respectively. Quantification by peak height measurement was performed with a Model TR-250 Toricorder (Tokyo Rikokikai Co. Ltd., Japan). The eluate was chloroform (sat. H2O) : n-hexane : acetonitrile : methanol ( 750/150/38/0.5, v/v/v/v ). The flow rate was 2 ml min-1. The detection limits of the HPLC system for aflatoxins B1, B2, G1, and G2 were 1.9, 2.5, 0.1, and 0.1 ng g-1, respectively.


An average of 54.8 and 58.9% of beans from Ontario and Taiwan, respectively, had seedborne fungi, of which 22.8 and 24.5%, respectively, had visible signs of fungal infection. The seedborne fungi and their frequencies of isolation are summarized in Tables 1, 2, and 3. The number of fungal genera and species isolated was 24 and 36, respectively, for Ontario beans, and was 28 and 41, respectively, for Taiwan beans. Among the 24 genera found in the Ontario bean lots, the most-frequently isolated ones were Alternaria (61.1%), Fusarium (18.0%), Rhizoctonia (6.1%), Penicillium (5.2%), Rhizopus (3.2%), Sclerotinia (3.0%), Gliocladium (2.2%), and Mucor (1.7%). Among the 36 genera found in the bean lots from Taiwan, the most-frequently isolated ones were Aspergillus (48.5%), Penicillium (27.6%), Eurotium (6.7%), Rhizopus (5.3%), and Curvularia (2.4%). Other isolated genera, which had a frequency of isolation of less than 1%, were mainly saprophytic fungi and several minor bean pathogens. With such a low occurrence, they might have little consequence in bean quality and mycotoxin production.

Both the healthy and the infected bean samples were analyzed for fusarium toxins and aflatoxins. In the healthy samples from Ontario and Taiwan, no fusarium toxins or aflatoxins were detected. In the infected bean samples, no aflatoxins were detected in the samples from Ontario and no fusarium toxins were detected in the samples from Taiwan. Fusarium toxins contained in the infected Ontario bean samples averaged 9.2 µg g-1 DAS, 6.5 µg g-1 DON, 5.5 µg g-1 T-2, and 1.8 µg g-1 FB1. In the infected samples from Taiwan, however, aflatoxins B1, B2, G1, and G2 were detected in concentrations averaging 0.23, 0.16, 20.37 and 0.72 µg g-1, respectively. All analyses were performed by TLC and subsequently confirmed by GC or HPLC.


Taiwan produced bean has a higher percentages of seedborne fungi and fungal infection than does Ontario produced bean. This is attributable to the fact that conditions for fungal growth and infection are more favorable in Taiwan than in Ontario. Taiwan has a subtropic climate, which is characterized by high precipitation and temperature, while Ontario has a temperate, semi-arid climate. Thus, the average temperature, precipitation, and humidity during the bean growing season in Taiwan are consid

erably higher than those in Ontario. Anderson (1985) and Miller and Roy (1982) found that seed lots of soybean produced in Ontario had fewer mycoflora than those produced in Mississippi, USA.

The number of beans with seedborne fungi was twice the number of those with visible fungal infection. This was true to both Ontario and Taiwan produced bean. Thus, about 50% of beans with seedborne fungi were merely surface contaminated and/or superficially infected and were not distinguishable from uninfected beans with the naked eye. When these beans were analyzed, neither

Table 1. Percentages of fungi most-frequently isolated from diseased dry bean seeds (Phaseolus vulgaris) produced in Ontario, Canada.

Fungus Incidence of isolation (%)

Genus Species

Alternaria 61.1

A. alternata 55.8

A. solani 3.1

Alternaria spp. 2.2

Fusarium 18.0

F. culmorum 3.2

F. graminearum 3.8

F. moniliforme 2.1

F. oxysporum 2.6

F. solani 3.2

Fusarium spp. 1.0

Gliocladium virens 2.2 2.2

Mucor spp. 1.7 1.7

Penicillium 5.2

P. chrysogenum 3.2

P. viridicatum 1.1

Penicillium spp. 0.9

Rhizopus stolonifer 3.2 3.2

Rhizoctonia solani 6.1 6.1

Sclerotinia sclerotiorum 3.0 3.0

Table 2. Fungi most-frequently isolated from diseased dry bean seeds (Phaseolus vulgaris L.) produced in Taiwan.

Fungus Incidence of isolation (%)

Genus Species

Aspergillus 48.5

A. flavus 11.8

A. flavo-furcatus 5.1

A. Japonicus var. aculeatum 3.1

A. niger 20.6

A. ostianus 6.2

A. tamari 1.7

Curvularia lunata 2.4 2.4

Eurotium crustatum 6.7 6.7

Penicillium 27.6

P. chrysogenum 16.0

P. commune 4.3

P. puberlum 2.2

P. viridicatum 5.1

Rhizopus 5.3

R. microporus 3.6

R. stolonifer 1.7

Botanical Bulletin of Academia Sinica, Vol. 36, 1995

Table 3. Fungi infrequently isolated (incidencd of isolation <1%) from diseased dry bean seeds produced in Ontario and Taiwan.

Ontario Taiwan

Aspergillus flavus Alternaria tenuis

Aspergillus sp. Actinomyces spp.

Botrytis cinerea Aureobasidium sp.

Cladosporium sp. Chaetomium cochliodes

Ceratocystis sp. Cladosporium chlorocephalum

Chaetomium cochliodes Cladosporium herbarum

Cochliobolus sp. Colletrichum truncatum

Colletotrichum truncatum Circinella muscoe

Macrophomina phaseolina Cunninghamella echinulatum

Mycelia sterilia Cunninghamella elegans

Phoma sp. Fusarium oxysporum

Phytophthora parasitica Fusarium solani

Pythium ultimum Fusarium spp.

Pythium sp. Geotrichum candidum

Saccharomyces sp. Gliocladium sp.

Trichoderma harzianum Hyalodenron sp.

Trichoderma viride Macrophomina phaseolina

Trichoderma sp. Mortierella sp.

Trichothecium roseum Mucor spp.

Mycelia sterilia

Neurospora crassa

Nigrospora sphaerica

Phoma spp.

Pythium sp.

Rhizoctonia solani

Trichoderma hamatum

Trichoderma spp.

Ulocladium sp.

fusarium toxins nor aflatoxins were detected. It can therefore be said that for field crop products from countries where storage and transportation facilities are adequate, the detection of mycotoxins can focus on only the visibly-infected beans.

The composition of the mycoflora of bean produced in different regions under different climatic conditions differs greatly, but in a given region it varies little from year to year (Anderson, 1985; Miller and Roy, 1982). A profile of mycoflora can be developed for each exporting country or region so that the most probable mycotoxin-producing mycoflora can be identified, thus facilitating the proper detection of mycotoxins.

Aspergillus, Penicillium, and Fusarium are the most important mycotoxin-producing fungal genera (Betina, 1989). Ontario-produced bean was predominantly infected with Fusarium and Alternaria, and the infection by Aspergillus and Penicillium was minimal (Tables 1 and 3). Mycotoxins related to Aspergillus and Penicillium do not appear to be a concern. Moreover, Alternaria spp. are known to cause only superficial infection (Tu, 1985), and grain harvested with a high percentage of Alternaria contamination was sound (Christensen and Kaufmann, 1969, 1974). The possible presence of fusarium toxins is therefore the major concern. Aspergillus and Penicillium were isolated at high rates (58.5 and 17.6%, respectively) from Taiwan-produced bean, and Fusarium at a low rate (<1%). Thus, the focus should be on aflatoxins.

Analyses of the infected bean samples from Ontario and Taiwan for fusarium toxins and aflatoxins confirmed our predictions. Fusarium toxins were detected only in Ontario-produced bean and aflatoxins in Taiwan-produced bean (Table 4).

Table 4. Quantification of aflatoxins and fusarium toxins in beans produced in Ontario and Taiwan by gas chromatograph and high performane liquid chromatograph.

Mycotoxin Bean sample

Origin of bean Group Kind Healthy Fungal-infected (µg g-1)

Ontario Fusarium toxin DAS NDa 9.2 ± 0.45b

DON ND 6.5 ± 0.35

T-2 ND 5.5 ± 0.24

FB1 ND 1.8 ± 0.11

Aflatoxin B1 ND ND




Taiwan Fusarium toxin DAS ND ND




Aflatoxin B1 ND 0.23 ± 0.03

B2 ND 0.16 ± 0.05

G1 ND 20.37 ± 1.80

G2 ND 0.72 ± 0.11

aND = Not detected.

bEach value is average ± SD, N =3.

DAS = diacetoxyscripenol, DON = deoxynivalenol, T-2 = T-2 toxin, and FB1 = fumonisin B1.

Tseng et al. — Mycoflora and mycotoxins in bean

The differences in the mycoflora and mycotoxins associated with beans from the two regions are attributable to the differences in climatic conditions, crop sequence, crop diversity, and degree of insect damage. Hill et al. (1985) pointed out that in warm, moist subtropic and tropic climates, Aspergillus and Penicillium species colonized grain before harvest in greater number than it did in cooler, temperate regions of the world. Also, in the tropics and subtropics, insect damage to grain is greater than in temperate regions. Lillehoj et al. (1976) reported that insect damage to corn before harvest enhanced Aspergillus infection and mycotoxin production. In Taiwan, rice, sugarcane, and tree fruits are the main crops. They are prone to Aspergillus and Penicillium infection. In Ontario, however, the crop variety is limited mainly to corn, wheat, soybean, and bean. Fusaria are the major pathogens of corn and wheat. Thus, the high population of Fusaria in the fields is a probable cause of high levels of fusarium infection in bean.

We suggest that monitoring mycotoxin in imported bean can be simplified by use of predetermined profiles of bean mycoflora for each exporting country or region. This will allow identifying the most-probable mycotoxins. This can also be applied to other field crops, such as corn and soybean.

In this research, no attempt was made to analyze Alternaria and Penicillium toxins. Some Alternaria toxins, in addition to their phytoxicity, are known potent teratogenic and/or carcinogenic agents (Scott et al., 1985) and several Penicillium toxins are potent carcinogens. Further research on the isolates from these two genera is needed to determine their mycotoxin-producing capability.

Acknowledgments. We acknowledge the financial support of the National Science Council of the Republic of China (Grant NSC84-2311-B-001-019) and the technical assistance of Ms L. C. Soo and Ms C. Y. Lu of the Institute of Botany, Academia Sinica, Taiwan, ROC.

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