Occurrence of Fungal Species and Mycotoxins from Decayed Sugarcane (Saccharrum officinarum) in Egypt.

Seventy-three fungal species belonging to forty-three genera were isolated from 40 samples of Saccharrum officinarum (collected from Naage-Hamadi canal in Qena Governorate, Egypt). Aspergillus, Trichoderma, Mucor and Pythium were the most common genera on the two isolation media. The dominant species of Aspergillus were A. niger, A. flavus, A. ustus, A. terreus and A. wentii. Some species were dominant on 40 g/l sucrose such as Aspergillus niger, A. flavus, Emericella nidulans, Trichoderma viride, Torula herbarum and Mamaria echinoeotryoides, while the dominant species on 10 g/l glucose were Mucor circinelloides, Aspergillus niger, Torula herbarum and Trichoderma viride. Mycotoxins including aflatoxins B1, B2, G1 and G2, zearalenone and diacetoxyscirpenol were detected in the examined samples of Saccharrum officinarum. The mycelial growth of A. flavus, A. niger, Fusarium moniliforme and Torula herbarum decreased with the increase in Dimethoate concentrations, although 25 ppm was less effective than the higher levels of the insecticide (75~200 ppm). Dimethoate stimulated the activity of Go-T in A. niger, F. moniliforme and T. harbarum, while the Go-T activity was inhibited in A. flavus with the Dimethoate treatments.

Sugarcane plant (Saccharum officinarum) is the predominant crop used in sugar production in the upper Egypt. Sugar present in the stem of S. officinarum represents the main source for fungal growth. Accordingly, the fallen stems in the canal or in the irrigation water during the harvest become a source of pollution for the water and other cultivated plants. The pollution effects depend on the mycoflora grown on the stem and on some environmental factors such as temperature. Mohawed et al. (2001) studied the seasonal fluctuations of soil and root surface fungi of sugarcane in the upper Egypt. They isolated 73 species and 5 varieties representing 33 genera using glucose-, cellulose-and sucrose-Czapek's agar media. Abdel-raheem (1999) examined the presence of fresh-water ascomycetes in various decayed plant parts of Eucalyptus rostrata, Phragmites australis and Phoenix dactylifera, collected from the river Nile in Egypt. In addition, El-Sharouny et al. (1999) studied the biodiversity and distribution of fungi on submerged wood in the river Nile and irrigation canals in the upper Egypt. Since some molds can produce toxic metabolites (mycotoxins), proliferation of the organisms represents a potential health hazard (Northolt et al., 1995). Therefore, detection of fungal contaminants is essential to ensure safe and high quality food (Bullerman, 1979).

On the other hand, insecticides have been used to control insects and play a significant role in increasing crop production. They commonly affect some of the non-target organisms such as microbial population ranging from inhibitory to stimulatory effects. Dimethoate is a broad-spectrum insecticide and commonly used in sugarcane to control the white fly (Anonymous, 1989). The effect of insecticides on fungal growth and enzymes activity was studied by different workers (Audus, 1960; El-Hissy and Abdel Kader, 1980; Abd-Elaah, 1993).

Materials and Methods

Sugarcane samples

Forty samples of sugarcane stems (S. officinarum) were collected during two seasons (winter and summer 2002) from the Naage-Hamadi canal in Qena Governorate in upper Egypt. Each sample was represented by 10 decayed stem parts. The samples were transferred directly to the laboratory for fungal isolation and toxin analyses.

Isolation and identification of fungi from S. officinarum

The stem-samples were washed with sterilized distilled water. Each stem was cut into segments (ca. 0.5 cm long) by knife, then each segment was cut into four equal parts. These segments were placed on the surface of two solidified media, glucose (10 g/l) and sucrose (40 g/l) Czapek's agar to which chloromphnicol was added as a bacteriostatic agent (Smith and Dawson, 1944). Five Petri plates of tested medium were used for each stem sample. Plates were incubated at 25℃ for one week. For the recovery of aquatic fungi, stem segments from the collected samples were placed in Petri-dishes; 12 cm in diameter (6 replicates). The segments in each Petri-dish were then covered with sterile distilled water (20 ml) and 12 sterilized sesame seeds were introduced into each Petri-dish, as employed by El-Nagdy (1986).

The growing fungi were identified, counted as numbers per segment. The identification of fungal genera and species was performed according to Raper and Thom (1949), Gilman (1957), Raper and Fennell (1965), Domsch and Gams (1972), Booth (1971), Pratt and Heather (1973) and Lund (1978). Fungal species recovered from stem samples were purified on suitable media such as glucose-peptone-agar, malt-extract-agar, potato-dextrose-agar, potato-dextrose-yeast-agar, sabouraud's-dextrose-agar and Czapek's-medium.

Mycotoxin extraction from S. officinarum

Fifty grams of decayed stems of each sugarcane sample were transferred to 500 ml Erlenmeyer flasks containing 150 ml of chloroform each and placed in a shaker (200 rpm) for 16 hours, then filtered through filter paper (Whatman No. 1). The chloroform extract was dried over anhydrous sodium sulphate. The remaining stem samples were dried at 50℃ over night, followed by re-extraction by 150 ml of 90% methanol-water.

Chemical detection of mycotoxins

Thin layer chromatography (TLC) technique was carried out using precoated with Silica Gel type 60, F254 (MERCK, Germany). Aflatoxins B1, B2, G1 & G2; ochratoxins A & B; sterigmatocystin; citrinin; T-2 toxin; diacetoxyscirpenol (DAS); zearalenone; moniliformin and fusarin C were used as standards. The developing solvent systems used were methanol - chloroform (v/v 3 : 97), ethyl acetate-hexane (v/v, 70 : 30), ethanol - chloroform (v/v, 5 : 95) and toluene - acetone - methanol (v/v/v, 50 : 30 : 20). The developed plates were then viewed under UV light (254 and/or 366 nm) and sprayed with reagents for identification according to Gimeno (1976) and Vesonder (1986).

Determination the effect of Dimethoate insecticide on mycelial growth and Go-T activity

The effect of the insecticide Dimethoate (O,O-dimethyl S-methylcarbamoylmethyl phosphorodithioate, IUPAC) on mycelial growth and Go-T (glutamic-oxaloacetic transaminase) activity were studied using most commonly occurring fungal species namely; Aspergillus flavus, A. niger, Fusarium moniliforme and Torula herbarum. Fifteen Erlenmeyer flasks (250 ml), each containing 50 ml Czapeck's-Dox liquid medium, were used for each fungus. Triplicate flasks served as control, in which media were amended with 25, 75, 150 or 200 ppm of the insecticide. Each flask was inoculated with 1 ml of the spore suspension obtained from seven days old cultures (Czapeck's-Dox medium) of the required fungus. The flasks were then incubated at 27℃ for seven days, after which the mycelial filtrates were collected from the flasks by Buchner filtration using hardened filter papers, washed several times with sterile distilled water and weighted. For determination of Go-T activity, ten milligrams of the fresh mycelia were mixed and homogenized with 1.0 ml phosphate buffer. The extracts were clarified by centrifugation for 15 min at 8000 ×g and then analyzed for Go-T as described by Reitman and Frankel (1957) using the transaminases kit (Quimica Clinica Aplicada S.A.).

Results and Discussion

The total fungal isolates obtained from 40 decayed sugarcane (S. officinarum) samples using glucose or sucrose as a carbon source were listed in Table 1. These results showed that numbers of fungi were greatest on 10 g/l glucose medium than 40 g/l sucrose medium, while, the number of species isolated on sucrose medium was more diverse than that recovered on glucose medium. In this respect, 26 species belonging to 18 genera were collected from the 40 samples of sugarcane on glucose-medium, while thirty-eight species belonging to twenty-six genera were collected on the sucrose-medium. A total of 48 fungal species belonging to 32 genera were isolated from the 40 samples of sugarcane on both glucose- and sucrose-media during winter and summer seasons. Sixteen out of these species were isolated on both tested media, while 10 species were obtained only on glucose medium and 22 species were isolated only on sucrose medium. The dominant genera on the two types of media were Aspergillus, Trichoderma, Mucor, Mammaria, Torula and Cephalosporium.

Table 1

Fungi isolated from sugarcane on 10 g/l glucose and 40 g/l sucrose Czapeck's agar media

TC: total counts, TC%: percentage of total counts, F%: frequency of occurrence.

*S=summer and W=winter.

Fungi recovered on glucose-agar medium

Twenty-six species belonging to eighteen genera were recovered from 40 decayed sugarcane samples in water canal on glucose-Czapek's agar at 25℃ (Table 1). Aspergillus was the dominant genus representing 95% of the samples constituting 45.8% of the total number of fungi. It was represented by 7 species of which A. flavus (8.78%), A. niger (30.4%), and A. ustus (4.4%) were of high occurrence. These Aspergillus species were also recovered from sugarcane leaves, stem, bagasse and juice by Higgy et al. (1977), Sandhu and Sidhu (1980), Sandhu et al. (1980), Olufolaji (1986), Sivanesan and Waller (1986), Muhsin and Abdel-Kader (1995), and Abdel-Hafez et al. (1995).

The remaining Aspergillus species were of moderate to rare occurrence on 20~5% of the samples. These species namely, A. awamori (1.1% of the total number of fungi), A. terreus (0.6%), A. wentii (0.4%) and A. japonicus (0.2%). Trichoderma, Emericella, and Torula were the second in occurrence. These were recovered from 85%, 75% and 90% of the tested samples and represented by high occurrence by 19.4%, 11.9% and 7.9% of the total fungal, respectively. Each was represented by one species namely, Trichoderma viride, Emericella nidulans and Torula herbarum. Colletotrichum dematium was of high occurrence, it appeared in 45% of the sugarcane samples and constituted 3.93% of the total fungal; this agrees with Brinker and Seigler (1991). Eurotium chevaliere, Mucor heimalis, Pythium intermedium and Pilobolus sp. were of come moderate occurrence (40%, 40%, 30% and 35% of the samples) and constituted 1.5%, 1.4%, 1.3% and 1.8% of the total fungi, respectively. Each of Gliomastix cerealis and Melanospora fallax were of low occurrence (20% of the samples), matching 1.0% and 0.6% of total fungi, respectively.

The remaining species were of rare occurrence (5~10% of the samples) constituting 0.7~0.1% of the total fungi. These species were Apodachlya brachynema, Achlya megasperma, A. americana, Allomyces macrogynous, Curvularia tetramera, Cunninghamella elegans, Moncilium mucidum, Synecephalostrum racemosum and Rhizopous stolonifer. Several researches reported that some strains of these fungi produced several toxic metabolites (Debeaupuis and LaFont, 1985; Charles et al., 1979; Stinson, 1985; Megalla et al., 1985; Leitao et al., 1989).

Fungal genera and species recovered on sucrose agar

Thirty-eight species belonging to twenty-six genera were recovered from 40 sugercane samples on 40 g/l sucrose-Czapek's agar at 25℃ (Table 1). Abdel-Hafez et al. (1995) isolated 46 species and 2 varieties belonging to 20 genera from 50 sugarcane juice samples on glucose-, sucrose- and cellulose-Czapek's agar at 28℃. The dominant genera were Aspergillus (8 species), Trichoderma (1 species), Mucor (4 species), Torula (2 species) and Cladosporium (1 species). They occurred in samples at rates 0.5~85.0% of the total samples investigated. In this respect, Abdel-Sater and Sabah Saber (1999) recorded that Aspergillus, Eurotium and Penicillium were the most common genera in dried raisins using 20% sucrose-Czapek's agar at 28℃. These results almost agree with the findings of Abdel-Sater and Ismail (1993), Megalla et al. (1985), Ismail (1993) and Aran and Eke (1987) who noted that Aspergillus and Penicillium were the most common in Egyptian and Turkish foodstuffs, respectively. Of the Aspergillus, the most dominant species were Aspergillus awamori, A. niger, A. ustus, A. flavus, A. versicolor, A. oryzae and A. terreus. Trichoderma (80% of the samples) was second to Aspergillus and was represented by one species namely, T. viride which constitutes 16.1% of the total count of the isolates. Mucor came third and it was represented by four species namely, M. circinelloides, M. heimalis, M. plumbeus, and M. racemosus constituting 0.2~7.6% of the total fungi recovered (Table 1). Torula was represented by two species namely, T. herbarum and T. grisea constituting 4.9% and 0.6% of the total fungi recovered, respectively. Cladosporium herbarum, Cunninghamella elegans, Saccharomyces spp., Fusarium moniliforme and Cephalosporium curtipes were of moderate occurrence, they comprised 1.25~3.49% of the total fungi recovered. The following genera namely, Curvularia tetramera, Mammaria echinoeotryoides, Penicillium luteum and Pythium intermedium were represented by low occurrence and constituting 1.1~2.3% of the total fungi recovered. The remaining genera and species were represented by rare occurrence (5.0~10.0%); with a frequency of 0.1~0.8%. Saccharomyces spp appeared only on the sucrose medium.

Seasonal fluctuation of the fungal species

The results given in Table 1 showed that 26 species appeared on glucose medium in both summer and winter seasons. The seasonal fluctuation of these species on glucose medium revealed that 9 species appeared in winter, 7 species in summer and 10 species in both seasons. On the other hand, 38 fungal species were recovered on sucrose medium in both summer and winter seasons. 19 species of them were isolated in winter, 10 species in summer and 9 species isolated in both seasons on sucrose medium. Generally, 48 fungal species were recovered from the two seasons on the two tested media. Nineteen out of these species were isolated only in winter and 12 species were isolated only in summer, while 17 species were isolated in both seasons. Seasonal fluctuations of fungi were also studied by El-Hissy (1979), El-Hissy et al. (1982) and Steciow (1998).

Mycotoxin production

Fusarium moniliforme produced zearalenone and diacetoxyscirpenol toxins (Table 2). These results are in agreement with those of Basch and Mircua (1992). Fusarium was recorded as zearalenone producer in Egypt (El-Maraghy, 1984; El-Kady and El-Maraghy, 1982; El-Maghraby and El-Maraghy, 1988; El-Maghraby et al., 1995). Thin layer chromatography analysis revealed the significant amounts of aflatoxins B1, B2, G1 and G2 in the tested samples produced by A. flavus (Table 2). These aflatoxins particularly B1 are associated with acute poisoning of animal and human (Jukes, 1978), lack of appetite, weight loss, unthrifitness, neurological abnormalities, jaundice of mucous membrane, convulsions and death (Harwig and Munro, 1975), causes damage of chromosomes (El-Zawahri et al., 1977) and carcinogenic for human liver (Smith and Moss, 1985). Based on visual estimation, when the sugarcane samples were subjected to aflatoxin screening, A. niger, A. ustus and Emericella nidulans were not toxin producers. Similarly, the isolated Colletotrichum dematium was also not toxin producer. Brinker and Seigler (1991) isolated piceatannol as a phytoalexin from the infected sugarcane with Colletotrichum falcatum but not from healthy or wounded sugarcane.

Table 2

Visual estimation of mycotoxins in sugarcane samples

Effect of Dimethoate insecticide on mycelial growth and Go-T activity

Table 3, Figs. 1 and 2 indicate the effect of different concentrations of the insecticide Dimethoate on mycelial growth and Go-T activity in A. flavus, A. niger, F. moniliforme and T. harbarum. Generally, the growth of mycelium decreased with the increase in Dimethoate concentrations in all tested fungi. Although 25 ppm was less effective than the higher levels of the insecticide, highly significant decrease in mycelial fresh weight was observed at 75~200 ppm of Dimethoate in all studied fungi, as compared with the control treatment. At low level (25 ppm) of Dimetoate, mycelial growth decreased significantly in F. moniliforme and T. herbarum while the reduction in mycelial growth of A. flavus and A. niger did not statistically differ from the control treatment. The results revealed that the fungal growth decreased with the increase in pesticide concentrations, which comes in agreement with Abd-Elaah (1993) who found that Dimethoate sharply reduced the growth of Saprolegnia ferax, Achlya proliferoides and Dictyuchus sterilis. The effect of insecticides on the inhibition of mycelial dry weight of Aspergillus fumigatus and Fusarium moniliforme was also observed by El-Hissy and Abdel Kader (1980). They reported that the rate of inhibition to be also influenced by the type of the fungus, age of the mycelium and concentration of the pesticides.

Table 3

Mycelial fresh weight and GO-T content in the presence of different concentrations of Dimethoate

*, **: Significant and highly significant values as compared with the control treatment.

Fig. 1

The effect of Dimethoate on mycelial growth.

Fig. 2

The effect of Dimethoate on Go-T content.

The results indicated that the Dimethoate treatments stimulated the Go-T activity in A. niger and inhibited it in A. flavus, as compared with the respective control values (Table 3 and Fig. 2). The activity of Go-T in F. moniliforme was higher than that of the control at 25~150 ppm of the insecticide, while it was inhibited at the higher level (200 ppm). In T. herbarum, the lowest level of Dimethoate (25 ppm) greatly stimulated the Go-T activity, while its activity decreased with the increase of Dimethoate concentration. The above results revealed that the insecticide Dimethoate stimulated the activity of Go-T in A. niger, F. moniliforme and T. herbarum especially at low doses. The inhibitory effect was prominent in case of A. flavus, indicating that this fungus was more sensitive to this insecticide than the other tested fungi. Audus (1960) suggested that microorganisms can develop the ability of degrade pesticides either by enzyme induction or by mutation. Abd-Elaah (1993) found that the activity of Go-T increased in Saprolegnia ferax and Dictyuchus sterilis by the application of the insecticide Dimethoate and the herbicide Basta.