The antagonistic effects of Candida parapsilosis on the growth of Fusarium species and fumonisin production.

BACKGROUND AND
PURPOSE: Fusarium species are avid producers of secondary toxic and carcinogenic metabolites such as fumonisin. Contamination of food and feed products with fumonisin can be hazardous to the health of humans and animals and may lead to agricultural loss. Accordingly, in this study, we aimed to evaluate the effects of Candida parapsilosis on the growth and fumonisin production of Fusarium species.
MATERIALS AND METHODS: Mycelial growth rate of 26 Fusarium isolates, including F. verticillioides (n=6), F. proliferatum (n=18), F. solani (n=1), and F. oxysporum (n=1), in the presence of 42 C. parapsilosis strains was investigated by pour-plate method. The decline in fumonisin production was measured in co-cultured fungi in coarsely ground maize after four weeks of incubation in the dark at 22°C, using ELISA technique. For data analysis, paired t-test was performed, using SPSS version 20.
RESULTS: The mycelial growth and fumonisin production of Fusarium isolates significantly decreased in the presence of C. parapsilosis in comparison with the control cultures (P<0.05). The percentage of mycelial growth inhibition ranged from 56.36% to 74.54%. The minimum and maximum decline in total fumonisin production was 12% and 78%, respectively. F. oxysporum and F. solani were found to be minor fumonisin producers among the studied Fusarium species. On the other hand, a decline was reported in the growth of Fusarium species and fumonisin production in the presence of C. parapsilosis.
CONCLUSION: C. parapsilosis showed notable inhibitory activities against Fusarium isolates. Therefore, this fungal species could be considered as a biocontrol agent against the growth and fumonisin production of toxigenic Fusarium species in the future.

Introduction

Mycotoxins have been long recognized as a global problem for human and animal health. The threat only increases as the demand for available food supplies rises in response to world population growth [1]. Contamination of staple food grains by fungi has been reported in various regions, particularly in developing countries. This sort of contamination can happen at all stages of plant cultivation, i.e., pre-harvest, harvest, and storage stages.

The safety and nutritional quality of foodstuffs are often reduced by fungal toxins, which are metabolites produced by several fungal species, colonizing staple agricultural products and crops exported from developed and developing countries. Mold and mycotoxin contamination may be detected at any point of the supply chain. The climate of tropical and subtropical countries provides ideal conditions for mycotoxin production, which needs to be controlled by post-harvest processes, adequate equipments, and sound management practices [2].

Fusarium species constitute a group of fungi with a worldwide distribution. They are recognized as a common cause of contamination in various grains and a possible source of different mycotoxins, such as fumonisin, zearalenone, and trichothecene [3, 4]. Fumonisins are highly toxic carcinogenic metabolites, which are usually formed in plants, mainly infected with F. verticillioides or F. proliferatum. These metabolites are commonly found in maize or other agricultural products prior to harvest.

Food and feed stuff contamination with fumonisin is notable for the serious public health hazards and the associated economic burden [5-8]. This mycotoxin, which is structurally similar to sphingosine, interferes with the metabolism of sphingolipids and leads to cell apoptosis [9]. In the literature, leukoencephalomalacia, porcine pulmonary edema, hepatotoxicity, and hepatocarcinogenicity have been reported as the consequences of intoxication by fumonisin in horses, pigs, and rats, respectively [10- 13].

Today, fumonisin B1 is known as a risk factor for high rates of human esophageal cancer in South Africa and China [5-7, 14]. The same probably applies to Iran, as high rates of natural contamination with Fusarium strains have been reported in maize, wheat, and rice by Ghiasian et al. [15], Mohammadi-Gholami et al. [16], and Alizadeh et al. [8] in areas with major cultivation and high risk of esophageal cancer.

Use of biocontrol agents is an efficient and cost-effective strategy to control and decrease toxicity by fumonisin [17]. The inhibitory properties of different fungal and bacterial organisms on the growth and mycotoxin production of various toxigenic fungal species have been reported in several studies. In this regard, Turkel et al. [18] introduced Metschnikowia pulcherrima UM15 as a highly effective yeast against various Fusarium, Aspergillus, and Penicillium species. Similarly, Rocha et al. [19] showed the major suppressive effect of Bacillus thuringiensis on F. verticillioides growth and fumonisin production.

The antagonistic activities of M. pulcherrima strains against Candida tropicalis and C. albicans were reported by Csutak and colleagues [20]. Furthermore, the efficient inhibitory activities of Saccharomyces cerevisiae RC008 and RC016 strains against the growth and mycotoxin production of A. carbonarius and F. graminearum were reported by Armando and colleagues [17].

Niknejad et al. used C. parapsilosis as a biocontrol agent against the growth and aflatoxigenicity of Aspergillus species [21]. Considering the high toxigenicity of Fusarium isolates from different regions of Iran, as reported by Mohammad-Gholami et al. [16], control of these toxigenic fungi should be taken into account.

To the best of our knowledge, no study has been conducted on the role of C. parapsilosis as a biocontrol agent in decreasing fumonisin production and growth of Fusarium isolates. Accordingly, in this study, we aimed to evaluate the effects of C. parapsilosis on the growth and fumonisin production of Fusarium isolates.

Materials and Methods

A total of 42 clinical C. parapsilosis isolates and 26 clinical and environmental Fusarium isolates were evaluated in this study. All isolates were obtained from the culture bank at the medical mycology laboratory of School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. All fungal strains were stored in sterile distilled water. The working cultures were prepared from distilled water stocks after being transferred to Sabouraud Glucose Agar (SGA; Merck, Germany).

Cultures of C. parapsilosis on SGA were incubated at 30°C for 48 h and were used to prepare a suspension of yeast cells with a density of 106 cells/ml. Spore production by Fusarium isolates was induced on SGA plates, incubated at 28°C for 48 h. The spores were harvested in sterile water, containing 0.05% Tween 20, followed by vigorous agitation. Then, the mycelial debris was removed by filtration through sterile Whatman No. 1 paper. Conidial density was adjusted to 106 cells/ml, using a haemocytometer slide. The standard plate count method was used on SGA to confirm yeast and conidial viability [21].

C. parapsilosis Fusarium

In order to determine the inhibitory effects of C. parapsilosis on the growth of Fusarium isolates, 0.5 ml of each yeast suspension (106 cells/ml) was added to each plate (10 mm in diameter), containing 20 ml of molten SGA at 45°C. After solidification, the plates were centrally inoculated with 20 µl of conidial suspension of each Fusarium strain. After seven days of incubation at room temperature, colony diameters were measured using calipers.

The growing diameter of cultures, containing Fusarium and C. parapsilosis isolates, was compared with the control cultures (free of C. parapsilosis). For each colony, the two diameters measured at the orthogonal position were averaged to determine the mean diameter for each colony. All experiments were carried out with two separate replicate plates per treatment. The mean diameter of colonies was considered to be indicative of evolution. Afterwards, C. parapsilosis isolate with the most significant inhibitory effect on growth was selected by evaluating its inhibitory effect on fumonisin production.

Fusarium isolates were prepared for mycotoxin production on autoclaved, ground maize, according to mycotoxin protocols with minor modifications [15]. Then, 20 g of coarsely ground maize was moistened with 20 ml of distilled water in 10 mm diameter plates and autoclaved at 121°C for 20 min over two consecutive days. Briefly, 1 ml of C. parapsilosis IPC24A inoculum (106 cells/ml) was cultured in the prepared maize medium. Afterwards, 1 ml of each Fusarium isolate suspension was inoculated in the center of the plates. The suspension was incubated in the dark at 22°C for four weeks; all tests were carried out in triplicate.

Fumonisin was extracted from 5 g of each maize medium, using 70% methanol and filtration through Whatman No 1 paper. Quantitative ELISA technique for the analysis of fumonisin was performed after extract dilution, using sterile distilled water, according to the manufacturer’s instructions in Ridascreen Fumonisin Kit (R-Biopharm, Germany). Considering the high concentration of toxins, further dilutions of the filtrate, i.e., 1:500 and 1:1000, were prepared prior to the assay, using distilled water. The results were multiplied by the dilution coefficient.

For statistical analysis, paired t-test was performed, using SPSS version 20. P-value less than 0.05 was considered statistically significant.

Results

The statistical analysis of the results revealed a significant difference in colony diameter and fumonisin production between co-cultured Fusarium and C. parapsilosis isolates and the control cultures (Fusarium isolates alone) (P<0.05). The mean colony diameter of the control cultures and co-cultured isolates was 56.7 mm and 19.8 mm, respectively (Figure 1).

Figure 1

The inhibitory effects of Candida parapsilosis on the growth rate of Fusarium proliferatum after seven days of incubation at room temperature; a) Fusarium proliferatum, b) Fusarium proliferatum in the presence of Candida parapsilosis

Overall, C. parapsilosis strains were able to decrease the growth of all Fusarium isolates. However, C. parapsilosis IPC24A isolated from infected nails was found to be the most effective strain, decreasing the colony diameter of Fusarium isolates; therefore, this strain was used throughout the study. In the present study, the difference between decreased growth and fumonisin production among the studied Fusarium species was insignificant. The detailed results on colony diameter and amount of fumonisin production by Fusarium isolates are presented in Table 1.

Table 1

The mean colony diameter and amount of fumonisin produced by Fusarium species before and after treatment with Candida parapsilosis IPC24A

Isolate	Species	Mean colony diameter (mm)before treatment	Colony diameter (mm) after treatment(reduction%)	Fumonisins (ppm)before treatment	Fumonisins (ppm)after treatment(reduction%)
1M	F. proliferatum	60	19 (68.33)	310	200 (35.5)
2M	F. proliferatum	57	22 (61.4)	200	120 (40)
3M	F. proliferatum	55	24 (56.36)	2000	1750 (12.5)
4M	F. proliferatum	55	18 (67.3)	2000	1600 (20)
5M	F. proliferatum	60	14 (76.7)	220	148 (32.72)
6M	F. verticillioides	58	19 (67.24)	2000	1670 (16.5)
7M	F. verticillioides	60	23 (61.7)	1450	900 (37.93)
8M	F. proliferatum	55	22 (60)	2100	1270 (39.52)
9M	F. proliferatum	55	17 (69)	370	296 (20)
10W	F. proliferatum	57	22 (61.4)	0.2	0.14 (30)
11M	F. proliferatum	60	23 (61.7)	2160	1620 (25)
12M	F. proliferatum	54	16 (40.4)	600	365 (39.16)
13M	F. proliferatum	55	16 (70.9)	1800	1404 (22)
14W	F. proliferatum	52	15 (71.1)	140	100 (28.57)
15W	F. proliferatum	55	21 (61.82)	970	737 (24)
16W	F. proliferatum	55	22 (60)	2000	260 (87)
17W	F. proliferatum	55	14 (74.54)	130	79 (39.23)
18M	F. verticillioides	53	15 (71.7)	200	123 (38.5)
19M	F. verticillioides	60	23 (61.7)	2000	1760 (12)
20M	F. verticillioides	58	21 (63.8)	2970	1200 (59.6)
21C	F. verticillioides	55	16 (70.9)	970	580 (40.2)
22C	F. proliferatum	60	18 (70)	130	96 (26.15)
23C	F. proliferatum	58	18 (68.9)	730	475 (34.93)
24C	F. proliferatum	54	23 (57.4)	2100	1650 (21.42)
25C	F. solani	58	20 (65.5)	130	90 (30.76)
26C	F. oxysporum	60	20 (66.7)	140	85 (39.3)

M: Maize, W: Wheat, C: Clinical, mm: Millimeter, ppm: Parts per million

Discussion

Cereal crops such as maize and wheat constitute an important part of human food and animal feed. It is estimated that 25% of food crops are contaminated with mycotoxins, produced by toxigenic fungal contaminants [22]. According to the Food and Drug Administration (FDA) of USA, the maximum acceptable level of total fumonisin is 2-4 ppm in corn products consumed by humans [23].

According to the International Agency for Research on Cancer, fumonisin is probably carcinogenic to humans; consequently, it is categorized in group 2B of carcinogens [8, 24-27]. Considering the toxic and potential carcinogenic properties of this mycotoxin, it is necessary to apply cost-effective and technically feasible methods to decrease or remove this hazardous compound [28].

Biological control of phytopathogens using microorganisms has been known as an effective method [17]. Yeasts are among the most efficient microorganisms, used as biocontrol agents. The inhibition of growth and mycotoxin production is generally attributed to the competition between other microorganisms and toxigenic filamentous fungi for nutritional, space, and other requirements [17].

In a study conducted by Armando et al., Saccharomyces cerevisiae was able to inhibit the growth of A. carbonarius and F. graminearum and prevent the production of ochratoxin A, zearalenone, and deoxynivalenol [17]. This ability of S. cerevisiae is speculated to be related to the strain-dependent property of mycotoxin absorption [29]. Therefore, fungi by absorbing mycotoxin lead to the reduction or removal of hazardous compounds. The beta-glucan from the cell wall of S. cerevisiae is a probable involved compound in toxin absorption [21].

On the other hand, Csutak et al. reported competition for iron as the major mechanism of antagonistic action in M. pulcherrima strains against C. tropicalis and C. albicans [20]. Matic et al. [30] in a previous study investigated the efficacy of M. pulcherrima, Pichia guilliermondii, and P. anomala as biocontrols against F. fujikuroi. They proposed β-1,3-glucanase, fungicides or fungistatic compounds (such as ethanol and ethyl acetate), and hydrolase enzyme secretion as effective mechanisms of M. pulcherrima, P. anomala, and P. guilliermondii, respectively. Based on these findings, the active role of C. parapsilosis in our study might be due to one or a combination of these mechanisms.

In the present study, all Fusarium isolates from wheat and maize, as well as the clinical isolates, were fumonisin producers. Although F. oxysporum is recognized as a non-fumonisin producer species [31], our results revealed fumonisin production by this Fusarium species. The co-occurrence of other Fusarium toxins including nivalenol, zearalenone, and deoxynivalenol (DON) with other DON derivatives might be a contributing factor, owing to the relative cross-reactivity.

On the other hand, Rheeder et al. introduced F. oxysporum (in section Elegans) as a fumonisin-producing species [32]. In the present study, C. parapsilosis exhibited inhibitory effects against all Fusarium isolates. The growth rate and fumonisin production were significantly lower in co-cultured isolates in comparison with C. parapsilosis-free cultures. In addition, Niknejad et al. [21] reported the antagonistic effects of C. parapsilosis on mycelia growth and aflatoxin production by Aspergillus species .

Bacon et al. reported the antagonistic effects of Trichoderma species on the growth and fumonisin production of F. moniliforme in comparison with the control cultures [33]. Furthermore, the role of P. anomala in Penicillium verrucosum as an inhibitor of ochratoxin A production has been suggested in the literature [17]. A. flavus, A. niger, and A. ochraceus have been shown to have the ability to reduce fumonisin production by Fusarium species and destroy fumonisin [34]. Similar to the present results, the efficacy of some fungal species as biocontrol agents was confirmed in the mentioned study.

Almost all detoxification methods have some limitations. An ideal approach should be cost-effective, practical, and free of side-effects for humans and animals; therefore, combined application of different methods may be required. Also, unfavorable conditions for mycotoxin production should be considered in the storage period.

Conclusion

The antagonistic properties of C. parapsilosis strains are of great significance. Considering the opportunistic and pathogenic nature of this fungus, further research is required on the extracted fractions, effective components, and genes of this fungus for future use as efficient biocontrols against the growth and mycotoxin production of Fusarium species. In the present study, the ability of Fusarium isolates from different species and sources (particularly clinical isolates) to produce fumonisin was noteworthy.