Natural occurrence of mycotoxins in broad bean (Vicia faba L.) Seeds and their effect on Rhizobium-legume symbiosis

Soil Biof. Biochem. Vol. 26, No. 8, pp. 1081-1085, 1994 Copyright Q 1994 Elsevier Science Ltd Rinted in Gnat Britain. All rights reserved 003&0717(94)Eoo120 0038-0717/94 $7.00 + 0.00

NATURAL OCCURRENCE OF MYCOTOXINS IN BROAD BEAN (VICIA FABA L.) SEEDS AND THEIR EFFECT ON RHIZOBIUM-LEGUME SYMBIOSIS A.-L. E. MAHMOUDand M. H. AED-ALLA+ Department of Botany, Faculty of Science, Assiut University, Assiut, Egypt (Accepted 30 December 1993) Sumnnuy-Seeds of faba bean (Vicia faba L.) cultivar Gixa 3 were screened for the presence of mycotoxins. Eleven out of 100 samples were positive. Aflatoxins B, and B, were found in 7 samples with a mean concentration of 30 pg kg- ’ seeds.Afiatoxins B,, B,, G, and G, and ochratoxin A were each detected twice in separate samples with a mean concentration of 25 and 2Obg kg-‘, respectively. Mycotoxins at concentrations of 100 or 200 pg kg-’ soil significantly decreased nodule number, nodule fresh weight and total nitrogenam activity. This was translated into reductions in dry matter accumulation and nitrogen yield of the bean. Mycotoxins also suppressed specific nitrogenase activity, NADH-dependent glutamate dehydrogenase (NADH-GDH) as well as glutamate synthase (NADH-GOGAT) activities. In addition, mycotoxins inhibited synthesis of leghaemoglobin, carbohydrate and protein in the nodule cytosol. Of the mycotoxins tested, aflatoxin B, was the most toxic. The decline in nitorgenase activity and total N concentration in the plants could be attributed to mycotoxins interfering with normal nodule physiology and function.

INTRODUCTION

Mycotoxins can either be introduced artificially or they may possibly be produced naturally in soil. They are introduced into the soil when corn grain, contaminated with mycotoxins (more than 20 ng g-i grain) is ploughed into the soil (Rodricks, 1977; Ayres and Brooks, 1979). They are produced naturally in the soil by indigenous soil fungi capable of degrading a wide variety of organic compounds (Bell and Crawford, 1967). High concentrations of mycotoxins might occur in soil in loci where there is enough degradable organic matter (legume residues) to support abundant growth of mycotoxin-forming fungi. Indigenous soil fungi have frequently been demonstrated to be antagonistic to various species of Rhizobium (Chhonkar and Subba-Rao, 1966; Sethi and Subba-Rao, 1968). The production of mycotoxins by these fungi is primarily responsible for the antagonistic effects towards Rhizobium although competition, predation and parasitism may also be important (Chowdhury, 1977). In addition, mycotoxins may be formed in seeds by toxigenic mould invasion and are thus deposited in soil during planting. The natural occurrence of mycotoxins in broad bean (Viciu faba L.) seeds were reported by Girgis et al. (1977) and Saber (1992). The occurrence and toxicity of mycotoxins in agricultural products consumed by man and animals

*Author for correspondence.

have received considerable attention. Less attention, however, has been given to the ecological role of mycotoxins in soil where the influence of mycotoxins is unknown (Angle and Wagner, 1981). Mycotoxins inhibited the growth of soil microorganisms (Angle and Wagner, 1981), however, their effect on the Rhizobium-legume symbiosis was unknown. Our objective was to investigate the effect of different mycotoxins on nodulation, nitrogen-fixation and plant growth of faba bean. Leghaemoglobin, protein and carbohydrate contents of the nodules as well as glutamate dehydrogenase (GDH) and glutamate synthase (GGGAT) activities were also assessed. MATERIALS

AND METHODS

Source of broad bean seeds (cv. Giza 3) One hundred samples of seed (100 g) were collected from the seed stores of the Ministry of Agriculture at different localities in Egypt. Mycotoxin analysis

Each sample (25 g) was defatted by extraction with n-hexane for 10 h using soxhlet-type extraction. The defatted residue was extracted for another 10 h with chloroform. The chloroform extract was dried over anhydrous sodium sulphate, filtered then reduced under vacuum to near dryness. The residue was diluted with chloroform to 1 ml. The chloroform solution was analysed for the presence of the mycotoxins: aflatoxins (B,, Br, Gi and GJ, citrinin, zearalenone, T,-toxin, sterigmatocystin,

1081

1082

A.-L. E. MAHMOUDand

ochratoxin A, penicillic acid and patulin by thin-layer chromatography (Gimeno, 1979). Ailatoxins were confirmed on plates by treating with trifluoroacetic acid. After development in CHCl,-acetone (85-15), plates were examined under longwave U.V. radiation. AfIatoxin content was determined spectrophotometrically at 350 mn (A.O.A.C. 1975). Ochratoxin A present in the chloroform extracts was confirmed according to Scott et al. (1972) and quantified as described by Damoglou et al. (1984). Plant culture and experimental

conditions

Freshly-harvested faba bean seeds (Vicia fuba L. cv. Giza 3) were inoculated with Rhizobium leguminosarum RCR 1001 biovar viceae and planted into plastic pots (17 cm dia. x 22 cm depth). Each pot filled with 3 kg autoclaved clay soil treated with aflatoxin B,, ochratoxin A or kojic acid. Some physical and chemical characteristics of the soil were described by Abd-Alla (1992a). Mycotoxin

treatments

Two of the mycotoxins, aflatoxin B, and ochrotoxin A, which occur naturally in broad bean seeds as well as kojic acid, another toxic fungal metabolite (Clevstrom et al., 1989) were tested separately. Aflatoxin B, was kindly provided by Dr 0. L. Shoetwell, U.S. Department of Agriculture, Northern-Regional Research Laboratories, Peoria, III, U.S.A. and ochratoxin A by Dr R. D. Wyatt, Department of Poultry Science, North Carolina State Univeresity, N.C., U.S.A. Kojic acid had been isolated from A. fluvus (Megalla et al., 1987) and a sample was kindly provided by Professor Dr S. E. Megalla, Botany Department, Assiut University. Aflatoxin B,, ochratoxin A or kojic acid separately added to the soil at concentrations of 100 or 200 p g kg - ’ air dry soil. To do this an appropriate amount of a mycotoxin-methanol solution was added to the soil. The methanol was removed by evaporation and the mycotoxin-treated soil was thoroughly mixed. Broad beans were grown under standard conditions (Abd-Alla, 1992b). Seedlings were thinned to 2 per pot after 5 days. Plants grown in soil not treated with mycotoxins were used as a control. Treatments were arranged in a randomized block design with 3 replicates each. The experiment was done twice and the plants were harvested 60 days after planting. At this time, samples were large enough for chemical analysis of nodules. Acetylene

reduction assay

Nitrogenase activity was determined on a detached root system in a closed system as described by Abd-Alla (1992b) using the spectrophotometric method of LaRue and Kurz (1973). Then, the nodules of each individual root were counted and nodule fresh weights measured. Nitrogen content of the plant

M. H. &-ALLA

tissues were determined (Bremner, 1965). Preparation

by the Kjeldahl

method

of cell-free extracts of nodules

Excised nodule samples (500 mg) were extracted at 0-3°C in a hand-ground glass homogenizer with 5 ml extraction buffer containing 150 PM phosphate buffer (pH 6.9), 1.2m~ sucrose, ~PM EDTA and 3 PM cystein (Becana et al., 1985). The resulting homogenates were centrifuged at 3500g for 8 min to remove nodule debris. The supematant was centrifuged at 12,OOOg for 20min to sediment bacteroids. The supernatant of the second centrifugation, referred to as nodule cytosol, was assayed for plant enzyme activities, soluble proteins, carbohydrate and leghaemoglobin. Protein contents of nodule cytosol and bacteroids were determined according to Lowry et al. (1951). For the determination of water-soluble carbohydrates, the phenol-sulphuric acid method was used (Dubois et al., 1956). Leghaemoglobin was measured by calorimetry as described by Johnson and Hume (1973). Enzyme assay

The NADH-dependent glutamate dehydrogenase (NADH-GDH), and glutamate synthase (NADHGOGAT) activities were assayed spectrophotometritally following the oxidation of NADH (Bullen, 1956). The assay mixture consisted of 40 mru phosphate buffer (pH 8). Substrate concentrations for NADH-GDH assays were 100 PM NADH, 2.5 mM a-ketoglutarate, and 200 PM (NH4),SO,. NADHGOGAT activity was measured using 100 PM NADH, 2.5 mM cr-ketoglutarate and 10 mM L-glutamine. The reaction was begun by adding the enzyme preparation, and the absorbance was recorded continuously for 5 min at 340 nm in a double beam spectrophotometer (Spectronic 2000, Bausch & Lomb). Statistical

analysis

Statistical analysis was made by means of oneway analysis of variance (PC-state computer program). Means were separated by using the Duncans multiple-range test. RESULTS AND DISCUSSION

Natural samples

occurrence

of mycotoxins

in broad

bean

Eleven out of 100 samples of broad bean seeds, were contaminated naturally with aflatoxins (B,, B,, G, and G2) and ochratoxin A. Aflatoxins B, and B2 were found in 7 samples while aflatoxins B,, B,, G, and G, and ochratoxin A were each detected twice in separate samples (Table 1). These data agree with those of Saber (1992) who reported that broad bean seeds were naturally contaminated with aflatoxins (B,, B,, G, and G2) and confirm those of Scott et al. (1972) who detected ochractoxin A in white beans.

Mycotoxins and Rhizobium-legume symbiosis

1083

Table 1. Natural occurrenceof mycotoxinsin broad bean (Vicia f&a L. cv. Giza 3) seeds

Mycotoxin

Positive

Mycotoxin

Mean

sit&es out

concentration

concentration*

ci loo

detected

Aflatoxins B, and B,

7

Aflatoxins B,, B,, G, and G, Ochratoxin A

2 2

bsb-‘)

(mkg-‘)

33, 26, 29, 31 32, 34 and 25 22 and 28 18 and 22

30 25 20

*Each value represents the mean of positive samples.

However, Saleha et al. (1982) found that chick-pea arietinum L.) samples were contaminated during storage with aflatoxin B, only. (Cicer

Nodulation

Data presented in Table 2 show that the mycotoxins, aflatoxin B,, ochratoxin A and kojic acid, significantly decreased nodule number. With the high dose of mycotoxin, the effect was more clear. Analysis of variance revealed that aflatoxin B, was the most toxic. Also, in mycotoxin-treated plants, total nodule weight plant - ’ was reduced and followed a similar pattern to nodule number. Nodule numbers gg ’ dry weight root generally show that the high dose of mycotoxins (200 pg kg-’ soil) had a more inhibitory effect on nodule formation than on root growth. To our knowledge there are no comparable data on the effect of mycotoxins on nodulation. Sethi and Subba-Rao (1968) observed that culture filtrates of several species of the fungi Aspergillus, Fusarium, Paecilomyces and Penicillium inhibited growth of Rhizobium japonicum on artificial medium. This could be attributed to mycotoxins excreted by these fungi. The presence of antagonistic fungi in soil has been demonstratred to reduce the nodulation of legume roots (Robison, 1945; Angle et al., 1981). Nodule activity

Mycotoxins significantly reduced both total (per plant) and specific nitrogenase activity of nodules (Table 2). The results obtained for NADH-dependent glutamate dehydrogenase (NADH-GDH) and glutamate synthase (NADH-GOGAT) activities of nod-

ules showed similar trends. The decline in nitrogenase activity was accompanied by a decline in GGGAT and GDH activities. The results suggest that the depression in total nitrogenase activity was due to mycotoxin reducing nodule formation and nodule fresh weights per plant. The involvement of mycotoxins in suppressing enzyme synthesis in different seeds has been described by Black and Altschul (1965) and Chatterjee (1988). In mycotoxins-treated plants, protein, leghaemoglobin and carbohydrate contents per unit mass nodule were significantly reduced with increased concentration of mycotoxin (Table 3). It is likely that the depression in specific nitrogenase activity was due to mycotoxins reducing the protein, leghaemoglobin and carbohydrate contents of nodules. There are no published reports dealings with the effects of mycotoxins on nodule activity. However, Truelove et al. (1970) demonstrated that aflatoxin B, interferes with protein synthesis in seeds by inhibiting the incorporation of amino acids into protein. It was also envisaged that mycotoxins bind to DNA and thus prevents RNA and protein synthesis (Crisan, 1973). Plant growth and nitrogen content

An inhibitory effect of mycotoxins on dry matter accumulation of shoots and roots was noticed with the lower concentrations of aflatoxin B, and ochratoxin A. Calculation of shoot-to-root ratios on a dry weight basis shows that the inhibitory effect of a low dose of mycotoxins on dry matter is equally partitioned between shoots and roots. However, at the

Table 2. Effect of mycotoxins on nodulation and nodule function of Viciafaba plants. Each value represents the mean of three replicates* Acetylene reduction (pm01 C,H, h-‘)

Nodules plant-’ Mycotoxin treatments (pg kg-’ soil) Control Ckhratoxin A 100 200 Aflatoxin B, 100 200 Kojic acid 100 200

Enzyme activity GDH GGGAT

Nodule No. g-’ dry wt root

Plant-’

g-’ nodule fresh wt

92a

5.15a

2.7a

28.4a

16.3a

107a

Fresh wt (g) I .90a

70b 9e

1.60b 0.16d

92a 34c

3.52b 0.30d.e

2.2b 2.oc

23.2b 8.lb

12.3~ 5.3f

45c 1Oc

0.8Oc 0.15d

54c 32~

1.3Oc 0.2Oc

I .63e

16.3~ 9.4d

9.8e 3.2g

IOla 25d

1.51b 0.67~

92a 35c

3.lOb I .2cd

2.1obc

25.la 11.2cd

14.8b 11.2d

No.

1.30f 1.80d

(pm01 NADH oxidized g-’ nodule fresh wt min-‘)

*Values in the same column followed by the same letter are not significantly different at the 5% level by Duncan’s multiple range test.

A.-L. E. MAHMOUDand M. H. AID-ALU

1084

Table 3. Effect of m~toxins on protein, ~~a~oglobin and carbohydrate contents of Yi& fiabanodules.Each values muresents the mean of three m~Iicates*

Protein Mycotoxin treatments (pg kg-’ soil)

concentration Cytosol 3 95a

Control Ochratoxin A 100 200 Allatoxin B, 100 200 Kojic acid 100 200

Bacteriod (mg g-2’;adule

&gbaemoglobin concentration

Carbohydrate umcentration

Cytosol

Cytosol

fresh weight) 1.98a

5.6Oa

2.92b 1.28g

2.11b 0.88e

1.32c 0.32e

2.80d 2.oOe

2.06d 1.56e

1.91.Z 0.93e

l.lld 0.42e

2.6Od 1.7oe

2.15~ I .46f

1.95~ l.OId

1.86b 0.52e

4.95b 3.4oc

*Values in the same column followed by the same letter are not si~ificaody 5% level by Duncan’s multiple range test.

different at the

Table 4. Effect of mycotoxins on dry weights and total nitrogen of Viciafaba plants. Each value represent the mean of three replicates* Mycotoxin treatments (pg kg-’ soil) Control Ochratoxin A 100 200 Aflatoxin B, 100 200 Kojic acid 100 200

Total nitrogen (mg plant-l)

Dry weight (g plant-‘) Shoot-to-root ratio

Shoots

Roots

Shoots

Roots

6.86a

1.16a

5.9

1OQa

12a

4.32b 1.96C

0.76b 0.26c

5.7 7.5

6Oc 22d

07bc 03de

4.75b 2.79~

0.83b 0.34c

5.7 8.2

57c 28d

06cd 02e

6.32a 4.24b

1.10a 0.69b

5.7 6.2

96b 55c

10b 06d

*Values in the same column followed by the same letter are not significantly different at the 5% levels of Duncan’s multiple range test.

high dose, the inhibitory effect was more pronounced on roots than on shoots. The nitrogen content plant - i was significantly decreased by the application of each of the three mycotoxins tested {Table 4). The declines in nodulation and nitrogen fixation in plants treated with mycotoxins were translated into significant reduction in dry matter production and nitrogen yield. Our results support the findings of Chhonkar and Subba-Rao (1966) and Angle et al. (1981). They found that antagonistic fungi reduced the nodulation of Trifolium alexandrinum and soybean and correspondingly decreased growth and N content of these plants. We believe that this is the first report of the effect of mycotoxins on nodule function and growth of broad beans. To diminish the inhibititory effect of mycotoxins on nodulation and nitrogen fixation, broad bean cultivars which are less susceptible for mycotoxin formation and rhizobial strains which are more resistant to mycotoxins should be selected.

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