In Vitro Activity of Neem (Azadirachta indica) Oil on Growth and Ochratoxin A Production by Aspergillus carbonarius Isolates

Aspergillus carbonarius is a saprobic filamentous fungus, food spoiling fungus and a producer of ochratoxin A (OTA) mycotoxin. In this study, the in vitro antifungal activity of neem oil (0.12% p/p of azadirachtin) was evaluated against the growth of six strains of A. carbonarius and the production of OTA. Four different concentrations of neem oil were tested in addition to three incubation times. Only the concentration of 0.3% of neem oil inhibited more than 95% of the strain’s growth (97.6% ± 0.5%), while the use of 0.5% and 1.0% of neem oil showed lower antifungal activity, 40.2% ± 3.1 and 64.7% ± 1.1, respectively. There was a complete inhibition of OTA production with 0.1% and 0.3% neem oil in the four strains isolated in the laboratory from grapes. The present study shows that neem essential oil can be further evaluated as an auxiliary method for the reduction of mycelial growth and OTA production.


Introduction
Members of the Aspergillus spp., among many other toxigenic fungi, have been found to have a strong ecological link with human food supplies [1]. They are often associated with food and animal of neem oil had a medium effect (59-71%). Although the regression analysis indicated significant linear dose-responses (Table 2), the data fit a more cubic polynomial model ( Figure 1).   Table 3. Neem oil concentrations of 0.3% and 0.1% had a significant effect on lag phase, increasing the time needed for each strain to reach the exponential phase. The regression analysis showed a significant polynomial trend model correlation of different neem oil concentrations with the lag phase (Table 4), and the cubic trend seemed to better fit the model (Figure 1). The effect of neem oil treatments on OTA production by six A. carbonarius strains assayed after 2, 7 and 10 days of incubation is shown in Table 5. There was a complete inhibition in OTA production with the addition of 0.1% and 0.3% of neem oil for the four strains isolated from grapes whereas the two reference strains assayed (FRR5690 and A2034) produced low levels of OTA (28.2 and 22.2 ng/g, respectively) at 10 days of incubation. The absence of OTA production was also observed at two days of incubation and 1% of neem oil for FRR5690 and RCG4 strains.
The overall treatment time showed an increase in OTA production as incubation time increased and the regression analysis indicated significant linear dose-responses (Table 6; Figure 1).
An increase in OTA production was observed at 0.5% and 1% of neem oil. These two concentrations stimulated the OTA production at the end of the incubation period in 116.8 ± 78.8% and 498.8 ± 385.4%, respectively.
Single factors (concentration of neem oil and incubation time) as well as two-way interaction had a significant effect on OTA production by A. carbonarius strains studied (p < 0.001) ( Table 6). Table 6. Outputs of the ANOVA with single-degree-of-freedom orthogonal polynomial contrasts for the effects of different concentrations (C) of neem oil on ochratoxin A (OTA) production of six A. carbonarius strains (ST) at three incubation times (T).

Discussion
The effect of natural or synthetic compounds on Aspergillus section Flavi species growth and aflatoxin production has already been described by some authors. Gowda, Malathi and Suganthi [33] studied the effect of some chemical and herbal compounds on the growth of other toxicogenic specie, Aspergillus parasiticus, and it was observed that neem oil at 0.5% had moderate anti-fungal activity (84% reduction vs. control), and at 0.2% and 0.1% a low antifungal activity, 52% and 36%, respectively. A lower percentage of reduction in fungal biomass (51%) was obtained by Zeringue and Bhatnagar [34] who studied the effects of neem leaf volatiles on submerged cultures of the same species. The contradictions between these results and the present study can possibly be explained by the different biochemical pathways that regulate the synthesis of the different mycotoxins produced by studied Aspergillus species and by the differences in the composition and therefore, the properties of each oil fraction. Razzaghi-Abyaneh et al. [35] agreed with those authors previously mentioned since they reported that neem leaf and seed extract can cause morphological alterations in the exposed mycelia, and then lead to cellular destruction.

Discussion
The effect of natural or synthetic compounds on Aspergillus section Flavi species growth and aflatoxin production has already been described by some authors. Gowda, Malathi and Suganthi [33] studied the effect of some chemical and herbal compounds on the growth of other toxicogenic specie, Aspergillus parasiticus, and it was observed that neem oil at 0.5% had moderate anti-fungal activity (84% reduction vs. control), and at 0.2% and 0.1% a low antifungal activity, 52% and 36%, respectively. A lower percentage of reduction in fungal biomass (51%) was obtained by Zeringue and Bhatnagar [34] who studied the effects of neem leaf volatiles on submerged cultures of the same species. The contradictions between these results and the present study can possibly be explained by the different biochemical pathways that regulate the synthesis of the different mycotoxins produced by studied Aspergillus species and by the differences in the composition and therefore, the properties of each oil fraction. Razzaghi-Abyaneh et al. [35] agreed with those authors previously mentioned since they reported that neem leaf and seed extract can cause morphological alterations in the exposed mycelia, and then lead to cellular destruction.
Sitara et al. [36] concluded that the ideal concentrations for the reduction of the Alternaria alternata growth was 0.1% and 0.15% of neem oil extracted from seeds. These results corroborate the ideal concentrations of 0.1% and 0.3% found in this study. On the other hand, Bhatnagar and McCormick [37], studied the effects of the neem leaf extracts at 1%, 5%, 10%, 20% and 50% (v/v) on growth of A. parasiticus and concluded that it had no significant alterations at the mycelial growth. These results were very similar to Zeringue and Bhatnagar [38], that evaluated the effects of neem leaf extract on Aspergillus flavus and found only 4-7% of growth reduction.
On the other hand, Zeringue, Shih and Bhatnagar [39] studied the effects of clarified neem oil on growth in submerged and plated cultures of aflatoxigenic Aspergillus spp. which resulted in an increase of 11-31% measured by mycelia mass. Garcia and Garcia [40] agreed with those authors previously mentioned since they reported that neem did not inhibit either growth or aflatoxin production by A. flavus and A. parasiticus.
The 0.1% and 0.3% concentrations of neem oil completely inhibited the production of OTA for the four strains isolated from grapes. This can be explained since none of the four wild strains at 0.1% and three strains (RCG1, RCG2 and RCG3) at 0.3% had reached the exponential growth phase. However, at 0.5% and 1.0% concentrations, all the strains, except RCG1, showed increased production of OTA; this possibly occurred because these concentrations inhibited less of the mycelial growth in all of the six strains assayed. Another possibility is that the presence of the neem oil and the AZ compound in high concentrations could lead to an exacerbated oxidative stress situation by the fungus and an increase in OTA production.
According to the previous data in literature [41] the sensitivity of Aspergillus spp. to oxidative status perturbations is closely related to the production of mycotoxins. Several publications [41,42] addressed the exact mechanisms included in regulating the development and secondary metabolism of many Aspergillus spp. The production of mycotoxins is triggered by oxidative stress; an increase in reactive oxygen species (ROS) levels can increase mycotoxins levels, noting this phenomenon as one of the defense mechanisms of fungal cells. The tolerance of A. flavus and A. parasiticus isolates to oxidative stress has also been shown to be correlated with their levels of aflatoxin production. Roze et al. [43] showed that conidia of isolates with higher levels of aflatoxin production also exhibited greater viability when cultured in ROS-amended medium.
Finally, another possibility is that higher concentrations (0.5% and 1.0%) of neem oil could have exceeded the solubility limits of the tested medium and the effective compounds did not have the same activity as in the lower concentrations [44,45].
These results are divergent to Bhatnagar and McCormick [37] who found that using 10% concentration of neem leaf extract reduces 98% of A. parasiticus aflatoxins production even if there is no inhibition of the mycelial growth. Allameh et al. [46] concluded that the concentration needed to reduce 90% of the aflatoxin production from A. parasiticus was 50% of neem leaf extract (v/v). This concentration was 150 times greater than the ideal concentration of neem oil found in this study. Razzaghi-Abyaneh et al. [35] also found a high reduction (91.3%) of aflatoxin production per µg of mycelia by A. parasiticus using 1.56% of neem extracts from seeds and leaf.
While many compounds and substances have been found to effectively inhibit fungal growth and aflatoxin production, others have stimulatory properties and affect the biosynthesis or bioregulation of aflatoxins, just like what happened with the utilization of 0.5% and 1.0% concentration of neem oil [47]. Nowadays, the information about action mechanisms of these compounds on Aspergillus species is limited, however it is possible to assure that the neem oil has important antifungal properties against A. carbonarius.

Conclusions
These findings clearly indicate the use of neem oil in low concentrations, such as 0.1% and 0.3%, is a good possibility as an auxiliary control method for mycelial growth reduction in Aspergillus carbonarius strains and the inhibition of ochratoxin A production. Mycotoxin contamination in food poses serious health hazards to animals and humans. Very few scattered reports are available on the effects of plant oils on growth of ochratoxigenic fungi. This study can contribute to the knowledge to develop effective anti-mycotoxigenic natural products for reduction of mycotoxigenic fungi and mycotoxins in foods.

Culture Medium
Commercial neem oil (Base Fértil Agrícola, Cravinhos, SP, Brazil) was used in this study. According to the manufacturer's certificate of analysis, neem oil was extracted from seeds and contained 0.12% p/p of azadirachtin (= 1200 ppm). Neem oil was added to the Czapek yeast extract agar (CYA, HiMedia Laboratories Pvt. Ltd., Mumbai, India) at final concentrations of 0.1%, 0.3%, 0.5% or 1.0% (v/v) at 45 • C. Plates containing CYA media without neem oil were used as control.

Inoculation and Incubation Conditions
Spore suspensions of the six A. carbonarius strains were obtained by scraping the surface of a 7-day-old colony cultured in 2% malt extract agar (MEA, HiMedia Laboratories Pvt. Ltd., Mumbai, India) and transferring the conidia to a tube containing 10 mL of sterile distilled water supplemented with 0.1% Tween 20. The solution was homogenized and read in a spectrophotometer (530 nm) to obtain a transmittance between 80% and 82%, which corresponds to 1-5 × 10 6 colony forming units per milliliter (CFU/mL). Then the Petri plates were needle-inoculated centrally with 10 µL of the spore suspension. The plates were incubated at 25 • C ± 2 for a maximum of two weeks.

Growth Assessment
Two perpendicular diameters of the growing colonies were measured daily until the colony reached the edge of the plate or for a maximum of two weeks. The percentage inhibition of diameter growth (PIDG) values were determined according to the equation as below: Diameter of sample − Diameter of control Diameter of control × 100 Growth rate (mm day −1 ) was calculated by linear regression of colony diameter against time for each strain at each set of conditions tested, and the time at which the line intercepted the x-axis was used to calculate the lag phase (h) in relation to isolate and essential oil. In all cases, the experiments were carried out with three replicates per treatment. The growth of fungal cultures containing different concentrations of neem oil was compared with that of the control culture that was grown with no EO.

Ochratoxin A Extraction from Culture
Ochratoxin A production was analyzed after 2, 7 and 10 days of incubation. The methodology proposed by Bragulat, Abarca and Cabañes [49] with some modifications was used. On each sampling occasion, three agar plugs were removed from different points of the colony and extracted with 1 mL of methanol. The mixture was centrifuged at 14,000 rpm for 10 min. The solutions were filtered (syringe filters, 17 mm, 0.45 µm, nylon membranes), evaporated to dryness, and re-dissolved in 200 µL of mobile phase (acetonitrile:water:acetic acid, 57:41:2) and the extracts injected into the high-performance liquid chromatography system.

OTA Detection and Quantification
The OTA production was detected and quantified by reversed phase in a high performance liquid chromatography system Hewlett Packard Serie 1100 (HP/Agilent, Santa Clara, CA, USA) with fluorescence detection (λ exc 330 nm; λ em 460 nm) using a C 18 column (Supelcosil ™ LC-ABZ, 150 × 4.6 mm, 5 µm particle size), connected to a precolumn (Supelguard ™ LC-ABZ, 20 × 4.6 mm, 5 µm particle size). The mobile phase was pumped at 1.0 mL/min. The injection volume was 100 µL and the retention time was around 4 ± 1 min. The detection limit of the analyses was 1 ng/g [50].

Statistical Analyses
Statistical analyses were conducted using PROC GLM in SAS program (SAS Institute Inc., Cary, NC, USA). The differences between growth inhibition percentage, lag phase and OTA production at different concentration levels of neem oil by six Aspergillus carbonarius strains at 2, 7 and 10 days were analyzed statistically by analyses of variance (ANOVA). The statistical models used in ANOVA considered the effects of the dependent variables and "strains" as a covariate within the different concentration levels of neem oil. The independence of the covariate was formally checked. Means were compared by Fisher's LSD test to determine the influence of the neem oil on the ecophysiology of the strains assayed [51]. Orthogonal polynomial contrasts were used to determine linear, quadratic and cubic responses to neem oil. The OTA production data contained some results of "not detected" (ND). That is, the OTA concentration was not detected above the detection limit (DL) of the used method. The actual concentration represented by ND is some value below the DL, however, the analytical method cannot determine whether the ND is truly zero or some unquantifiable value between zero and the DL. For this situation we used the substitution method, replacing the ND with the DL value (1 ng/g). In the cases of "not growth" (NG) results, we performed the substitution with zero.

Conflicts of Interest:
The authors declare no conflicts of interest.