ELSEWIER International Journal of

Food Microbiology 29 (1996) 193-199

Aflatoxin-producing strains of Aspergillus flaws in the mould flora of the different greenhouse

substrates for the cultivation of cucumber ( Cucumis sativus, L.)

J.A. Ruiz, A. Bentabol, C. Gallego, R. Angulo, I. Acosta, M. Jodral *

Departamento de Brornatologia y Tecnologia de 10s Alimentos, Facultad de Veterinaria, Unicersidad de Cbrdoba, AL.. Medina-Azahara 9, 14005 Grdoba, Spain

Received 13 June 1994; accepted 11 May 1995

Abstract

Fungai contamination in a greenhouse for production of cucumbers (Cucumis sativus, L.) was studied. 158 samples of water, sand, air, leaves and fruits were analyzed. 25 different genera were isolated, the most frequent ones being Pullularia, Aspergillus, Paecilomyces, Alternaria, Fusar-

ium, Rhizopus and Penicillium. Pullularia, Aspergillus, Paecilomyces and Penicillium were present in all types of samples studied.

Twenty-one strains of A. flauus were identified and only eight of those produced aflatoxins 1’ in vitro ‘1 .

Keywords: Cucumber; Cucumis satiuus; Greenhouse cultivation; Moulds; A. flavus; Aflatoxins

1. Introduction

Greenhouse cultivation has extended all along the Mediterranean coast during the past 25 years, especially in the province of Almeria, in south-east Spain, where it represents over 50% of the land devoted to this type of growing in Spain. The microclimate inside the greenhouse makes it an ideal environment for the production of vegetables. However, these conditions encourage the proliferation and growth of moulds

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194 J.A. Ruiz et al./lnt. J. Food Microbiology 29 (1996) 193-199

which not only bring about changes in the crops but in some cases can be considered a risk for production of mycotoxins.

A greenhouse forms an ecosystem composed of several micro-environments influenc- ing each other (G6mez et al., 1984). This study has been carried out to investigate the

mycoflora of various samples from a greenhouse for production of cucumbers. The

potential toxigenicity of the Aspergillus flaws strains isolated has been investigated.

2. Materials and methods

2.1. Characteristics of greenhouse

The greenhouses for production of cucumber (Cucumis sativus, L.) consist of

eucalyptus wood posts covered with plastic sheets held up by an overhead wire structure. They measure: 3 m in height, 30 m in width and 150 m in length and cover an area of approximately 4500 m2 which is the average size of the greenhouses in this area.

Environmental conditions all the year round range between 21.2”C average temperature and relative humidity of 69%, with a passive ventilation.

The ground is made up of three layers: earth, manure (l-2 cm) and, finally, sand

(5-10 cm).

2.2. Sampling

The fungal contamination of the greenhouse was examined for water, air, sand, leaves and cucumbers. Samples were taken on the day the seedlings entered the greenhouse

(day 0) and at 12, 24, 36, 48 and 60 days of growing. No samples of the cucumbers were taken until after 36 days. A total of 158 samples were analyzed: water (24), sand

(24), air (48), leaves (42) and cucumbers (20). With the aim of studying the possible influence of their location in the greenhouse on

the fungal contamination of each of the substrates, samples were taken in two different locations inside the greenhouse, i.e. 3 and 10 m away from the side walls. The sand samples were collected at ground level, the water samples directly from the irrigation hydrant and those of the air by the method of sedimentation in 9 cm-diameter Petri plates, in potato dextrose agar (PDA, Difco) medium and with one minute of exposure. The leaf and fruits samples were collected, at two different levels, i.e. 50 and 100 cm

above the ground.

2.3. Enumeration and identification of moulds

10 g of samples were homogenized in 90 ml sterile peptone (0.1% w/v) for 5 min using an MSE Homogenizer, (Venitron Medical Products, Multimix Lourdes, Mod. MM-1B) moulds were enumerated using duplicate surface spread counts (Jodral et al., 1993) on potato dextrose agar (PDA, Difco) incubated at 25°C for 5 days and at 37°C for 3-5 days, respectively.

J.A. Ruiz et al./Int. J. Food Microbiology 29 (1996) 193-199 195

Fungal colonies were identified according to Smith (1963), Raper and Fennel1 (1965),

Ainsworth et al. (19731, Alarca Salat (1980), Dragoni et al. (1980) and Fassatiova

(1986).

2.4. Screening for aflatoxin-producing strains of Aspergillus jlacus

Identification of isolates as A. jlar;us, in the sense of the series consisting of A.

jlavus Link and A. parasiticus Speare (Thorn and Raper, 1945), was based on colony colour and gross morphology of conidial heads (Raper and Fenell, 1965). These isolates

were tested for production of orange pigment on A. flaws and A. parasiticus agar (AFPA) (Pitt et al., 1983).

Production of aflatoxin was detected using aflatoxin production agar (APA) (Hara et

al., 1974). As APA appeared to be inhibitory to the germination of spores of some isolates (data not included), all APA plates were inoculated with 4 mm plugs cut from the margins of 4-day PDA plate cultures, and examined for fluorescence under UV light

(360 nm) after incubation at 25°C for 12 days.

2.5. Extraction of APA plates and detection of aflatoxins by thin-layer chromatography

The procedure of Rojas et al. (1991) was followed; 30 g agar medium from duplicate fluorescent plates were macerated in 50 ml distilled water and extracted in 50 ml

chloroform (purity 99%, Panreac). The mixture was centrifuged at 3000 X g for 10 min, the chloroform phase was poured off and the agar, together with the aqueous phase, was again collected in a Waring Blendor and extracted once more with 25 ml of chloroform.

The chloroform extracts were combined and concentrated to dryness at 55°C in a

nitrogen atmosphere. Thin-layer chromatography and aflatoxin quantification were carried out as described

by Garrido et al. (1992). The quantification of aflatoxins was carried out by the fluorimetric method. A Perkin-Elmer model 3000 spectrofluorometer was used. The primary filter used was 365 nm, the secondary filter was for B, and B, was 430 nm, and for G, and G2 it was 450 nm. The detection limits were 4-5 pg/kg of aflatoxins.

3. Results and discussion

The fungal contamination of the different samples from the greenhouse throughout

the cultivation of cucumbers is shown in Table 1. As seen from the Table, the highest mean contamination was formed on the leaves which may be due to their larger surface, their wrinkled, velvety structure and the fact that as they appear early and accumulate

spores for a longer period of time. As they are protected from the wind and rain, the contaminating fungi are not removed and affected as they would have been in the free land. The leaves also harboured the greatest variety of moulds, i.e. 25 genera (Table 2). This suggested that an analysis of fungal contamination in cucumber leaves could be used as a biological indicator of the evolution of mycological contamination in green- houses, thus avoiding unnecessary fungicidal treatments.

196 J.A. Ruiz et al./lnt. J. Food Microbiology 29 (1996) 193-199

Table 1

Fungal contamination of the different greenhouse substrates for the cucumber production

Samples Number of samples Mean a Range a

(n)

Water 24

Sand 24

Air 48

Leaves 42

Cucumbers 20

a Expressed as CFU/g.

b expressed as CFU/cm* min.

8.4 x 10’ 2.10*-3.104

1.2x lo4 6.10’-7.10”

3.0x 10 o- > 300 b

5.8 x lo4 O-39. lo4

1.4x 10’ o-1 104

Table 2

Frequency of occurrence (%) of different mold genera in the different samples from greenhouse for cucumber

production

Genera Samples

water sand air leaf cucumber (n = 24) (n = 24) (n = 48) (n = 42) (n = 20)

Absidia Altemaria Aspergillus Beauceria Botryosporium Botryotrichum Botrytis Byssochlamys Cladosporium Chrysosporium Doratomyces Epicoccum Fusarium Gliocladium Humicola Monilia Mucor Oidiodendron Paecilomyces Penicillium Phialophora Phoma Phycomyces Pullularia Rhizoctonia Rhizomucor Rhizopus Sporotrichum Syncephalastrum Stemphylium Verticillium

75 63

2 29 31 _

2 64 31 _

29 _ 4

2 12

_ 25 20

5 _

5 5

4 _ 4 _ _ - 42

8 4

_ _

_ _ 75 67 25 13

8 38

_ 50 100 50

_ 4 _ _

29

4

_

_ _

10

13

4 _

58

_ 19 _

4

2 _

2

5 45

7 5

12 2

33 14 5

19 5

62 7 5

26 2

5 2

10

_ 20 15

10

70 5 5

20 5

15

J.A. Ruiz et al./Int. J. Food Microbiology 29 (1996) 193-199 197

Table 3

Frequencies (%I of Aspergillus spp. isolated in the different samples from greenhouse for cucumber

production

Species Samples

Water Sand Air Leaves Crops Total

A. carbonarius

A. jischeri

A. jlavipes

A. ,jlaws

A. ,fumigatus

A. glaucus

A. nidulans

A. niger

A. ochraceus

A. oryae

A. terreus

A. rersicolor

A. ustus

A. wen tii

Total

_ _ 5.1 (2)

100 (18) 2.6 (1)

10.2 (4)

_ 2.6 (1)

_ 5.1 (2)

- 5.1 (2)

_ 10.2 (4)

_ 17.9 (7) _ _

_ 17.9 (7)

_ 7.7 (3)

_ 12.8 (5)

2.6 (1)

(18) (39)

5.3 (1)

_ 26.3 (5)

5.3 (1) _

10.5 (2)

31.6 (6)

5.3 (1)

10.5 (2)

5.3 (1)

4(l)

28 (7)

20 (5)

16 (4)

16 (4)

4(l)

12 (3)

(25)

1.9 (2)

_ 1.9 (2)

_ 0.9 (1)

25 (1) 33.3 (35)

25 (1) 2.8 (3)

25 (1) 2.8 (3)

_ 1.9 (2)

_ 10.5 (1)

25(l) 17.1 (18)

_ 1.9 (1)

_ 12.3 (13)

_ 3.8 (4)

5.7 (6)

3.8 (4)

(4) (105)

a No. of positive samples in brackets.

The most frequently isolated genus was Pullularia (50-100%) which was to be expected since it is a natural contaminant of all products containing humid cellulose (Smith, 1963). This genus was followed in frequency of appearance by: Aspergillus (20-75%), Paecilomyces (lo-75%), Penicillium, Alternaria, Fusarium, and Rhizopus.

Pullulariu, Aspergillus, Paecilomyces and Penicillium were the only genera present in all types of samples studied which indicated a possible relation between the different

parts of the ecosystem. No statistically significant qualitative-quantative differences were observed in the

Table 4

Production of aflatoxin in APA medium (Hara et al., 1974) of strains of A. Jams isolated from samples of

sandand leaves from a greenhouse for cucumber production

Strains Aflatoxin concentration ( fig/g agar) ’

B, B? G, G2 Sand- 1

Leaf- 1 Leaf-2

Leaf-3

Leaf-4

Leaf-5

Leaf-6

Leaf-7

2.32 0.24

77.37 5.66

traces traces

7.15 3.58

51.11 8.56

293.04 25.41

traces traces

traces traces

0.85

26.07

traces _

12.60

127.65

traces

traces

0.28

4.11

traces _

8.14

39.12

traces

traces

* 12 days of incubation.

198 J.A. Ruiz et al./Int. J. Food Microbiology 29 (1996) 193-199

fungal isolations either between the locations or between the heights at which the

different substrates were controlled (results not shown). Fourteen species (Table 3) of Aspergillus were isolated with A. jlavus being the

most frequent one (33.3%) and the only one present in all types of samples. It is followed by: A. ochruceus (17.1%), A. terreus (12.3%) and A. niger (10.5%).

Sand was the substrate in the which greatest number of species of Aspergillus were isolated and the leaves were those most contaminated by strains of A. jlavus.

Among the strains of A. flavus isolated 21 including eight from the leaves and from the cucumber (Table 4) grown in APA medium where 8 (38%) turned out to be

toxigenic; 7 toxigenic strains (87.5%) were isolated from the leaves. The A. flavus strains isolated were capable of synthesizing amounts of aflatoxins

which ranged from non-quantificable amounts to 293 ppb of aflatoxin B,; according to

Smith and Moss (1985) and Swanson (19871, these strains can be considered as possessing a high toxigenic capacity. This indicates a need for mycological control of cucumber and possibly other vegetables as well as the need for controlled storage

conditions.

Acknowledgements

The authors thank Ms. Gloria Femandez-Marin and Mrs. Carmen Cadenas for their

invaluable collaboration and analytical assistance during the development of this work.

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