Biotechnological methods are those in which biological systems or their derivates are used in order to obtain better products. From among them, talking about AF control, we can highlight the biological control, the use of natural extracts and essential oils and genetic engineering to mention a few.
An option to supplement, but not to supplant the traditional methods of AF control is biological control. Most AF biological control programs can truly be defined as biocompetition since they do not utilize parasites or diseases of the pest, but instead use atoxigenic Aspergillus species to competitively exclude toxigenic fungi . Augmentative biological control is as a pest management tactic that utilizes the deliberate introduction of living natural enemies to low the population level of invasive pests. Biological control has been utilized for more than 100 years in efforts to control a wide number of agricultural pests including fungi, insects and weeds . Biocontrol strategies have been implemented to control AF contamination in several important agricultural crops, such as peanut, cotton and corn [43, 45, 46]. Some authors have reviewed some biological methods using bacteria, yeasts and fungi as competitors for containment of A. flavus growth and/or toxin production [46, 47]. Natural population of fungi like A. flavus, consists of toxigenic strains that produce copious amount of AF and atoxigenic strains that lack the capacity to produce AF. In the competitive exclusion mechanism, introduced atoxigenic strains out compete and exclude toxigenic strains from colonizing grains thereby reducing AF production in contaminated grains . The use of A. flavus atoxigenic strains (afla–) reduce AF contamination in many crops; nevertheless, the mechanism by which a non-aflatoxigenic strain interferes with AF accumulation of toxigenic strains has not been definitively elucidated [49, 50].
Since the last decade of the past century, some yeasts and bacteria have shown to be effective on controlling fruits and vegetables postharvest diseases. In the early nineties, biological control of grain fungi was studied only to a limited extent. Most of the studies had dealt mainly with the interaction between mycotoxigenic strains (mostly aflatoxigenic ones) and other fungi, occurring naturally on grains, grown in competition. A limited number of fungi (especially Aspergillus niger van Tieghem), yeasts and bacteria were found to inhibit, detoxify or metabolize AF; however, it was determined that their antagonistic effect was highly dependent on cultural and environmental conditions . There has been found that the yeast Pichia guilliermondii is effective in controlling major citrus fruit rots . Based in those studies, in 1993, Paster and collaborators evaluated the efficacy of Pichia guilliermondii Wickerham for the control of the common Aspergillus flavus storage fungus and the natural microflora of soya beans, obtaining good results. The ability of Pichia guilliermondii to inhibit growth of grain microflora was studied using naturally contaminated soya beans and sterilized soya beans artificially inoculated with Aspergillus flavus. When A. flavus (at a spore concentration of 102 spores ml-1) and P. guilliermondii (at concentrations of 107 or 109 spores ml-1) were applied simultaneously to sterilized soya beans, fungal proliferation was inhibited during 16 days of storage. Application of yeast cells 3 days prior to fungal inoculation resulted in decreased inhibitory activity. The inhibitory effect of the yeast was compared with that of propionic acid using naturally infested soya beans at two levels of moisture content (11 and 16%). At both levels the yeast prevented fungal proliferation on the grain for a limited period, but propionic acid showed better fungistatic activity .
During 1994 and 1995, studies were conducted in the environmental control plot facility at the National Peanut Research Laboratory in Georgia to determine the effect of different inoculum rates of biological control agents on preharvest AF contamination of Florunner peanuts. Biocontrol agents were nontoxigenic color mutants of Aspergillus flavus and Aspergillus parasiticus that were grown on rice for use as soil inoculum. Those results were published three years later . Findings like these were the basis of further studies focused on the use of aflatoxigenic Aspergillus species that researchers are still investigating with more detail.
In recent years, some antagonists have been applied in biocontrol of postharvest diseases of agricultural products. Naturally occurring populations of atoxigenic strains are considered reservoirs from which to select strongest biocompetitors. The atoxigenic strains colonizing the environment where crops are affected by repeated AF outbreaks should have adapted to, and hence acquired, a superior fitness, for the relevant environment. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing AF production and/or to give a preliminary indication of strain performance when released in the field . The mechanisms by which afla– strains interfere with AF accumulation has not yet been definitively established. The prevalent opinion is that it depends on the competitive exclusion of AF producer (afla+) strains from the substrate as a result of (a successful) physical displacement and competition for nutrients by afla– strains. However, different hypotheses may still be taken into consideration .
Biological control is a promising approach for reducing both preharvest and postharvest AF contamination. There are some studies that report reductions in AF that are achieved by applying nontoxigenic strains of A. flavus and A. parasiticus to soil around developing plants, especially in peanuts. When late-season drought conditions make peanuts susceptible to invasion and growth by these fungi, the applied nontoxigenic strains competitively exclude toxigenic strains present in the soil and thereby reduce subsequent AF concentrations. Reductions in AF contamination with the use of nontoxigenic strains, has also been demonstrated in corn and cottonseed [56-59].
In 2003, Dorner and collaborators reported the results of a study that was conducted to evaluate the efficacy of three formulations of nontoxigenic strains of Aspergillus flavus and Aspergillus parasiticus to reduce preharvest AF contamination of peanuts during two years. Formulations included a solid-state fermented rice, fungal conidia encapsulated in an extrusion product termed Pesta and conidia encapsulated in pregelatinized corn flour granules. Analysis of soils for A. flavus and A. parasiticus showed that a large soil population of the nontoxigenic strains resulted from all formulations. In the first year, the percentage of kernels infected by wild-type A. flavus and A. parasiticus was significantly reduced in plots treated with rice and corn flour granules, but it was reduced only in the rice-treated plots in year two. There were no significant differences in total infection of kernels by all strains of A. flavus and A. parasiticus in either year. AF concentrations in peanuts were significantly reduced in year two by all formulation treatments with an average reduction of 92%. Reductions were also noted for all formulation treatments in year one (average 86%), but they were not statistically significant because of wide variation in the AF concentrations in the untreated controls. Each of the formulations tested, therefore, was effective in delivering competitive levels of nontoxigenic strains of A. flavus and A. parasiticus to soil and in reducing subsequent AF contamination of peanuts . The maize endophyte Acremonium zeae is antagonistic to kernel rotting and mycotoxin producing fungi Aspergillus flavus and Fusarium verticillioides in cultural tests for antagonism, and interferes with A. flavus infection and AF contamination of preharvest maize kernels. In 2005, Wicklow, reported results of chemical studies of an organic extract from maize kernel fermentations of Acremonium zeae (NRRL 13540), which displayed significant antifungal activity against Aspergillus flavus and F. verticillioides, and revealed that the metabolites accounting for this activity were two newly reported antibiotics pyrrocidines A and B. Pyrrocidines were detected in fermentation extracts for 12 NRRL cultures of Acremonium zeae isolated from maize kernels harvested in different places. Pyrrocidine B was detected in whole symptomatic maize kernels removed at harvest from ears of a commercial hybrid that were wound-inoculated in the milk stage with A. zeae (NRRL 13540) or (NRRL 13541). The pyrrocidines were first reported from the fermentation broth of an unidentified filamentous fungus LL-Cyan426, isolated from a mixed Douglas Fir hardwood forest on Crane Island Preserve, Washington, in 1993. Pyrrocidine A exhibited potent activity against most Gram-positive bacteria, including drug-resistant strains, and was also active against the yeast Candida albicans. In an evaluation of cultural antagonism between 13 isolates of A. zeae in pairings with A. flavus (NRRL 6541) and F. verticillioides (NRRL 25457), A. zeae (NRRL 6415) and (NRRL 34556) produced the strongest reaction, inhibiting both organisms at a distance while continuing to grow through the resulting clear zone at an unchanged rate. .
In 2005, Bandyopadhyay reported a test of twenty-four atoxigenic A. flavus isolates under field conditions in Nigeria to identify a few effective strains that could exclude toxigenic strains. These atoxigenic strains were evaluated for a set of selection criteria to further narrow down the numbers to a few for further use in biocontrol field experiments. Good criteria of selection will ensure that the candidate atoxigenic strains belong to unique vegetative compatibility groups (for which testers have been developed) that are unable to produce toxigenic progenies in the natural environment. Propensity to multiply, colonize and survive are other selection criteria to make sure that few reapplications will be required once the atoxigenic strains are introduced in the environment .
In 2006, Palumbo and collaborators isolated bacteria from California almond orchard samples to evaluate their potential antifungal activity against AF-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a neither nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented AF precursor norsolorinic acid (NOR) under conditions conducive to AF production. 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria .
Chang & Hua in 2007, from screening subgroups of nonaflatoxigenic A. flavus, identified an A. flavus isolate, TX9-8, which competed well with three A. flavus isolates producing low, intermediate, and high levels of AF, respectively. TX9-8 has a defective polyketide synthase gene (pksA), which is necessary for AF biosynthesis. Co-inoculating TX9-8 at the same time with large sclerotial (L strain) A. flavus isolates at a ratio of 1:1 or 1:10 (TX9-8:toxigenic) prevented AF accumulation. The intervention of TX9-8 on small sclerotial (S strain) A. flavus isolates varied and depended on isolate and ratio of co-inoculation. At a ratio of 1:1 TX9-8 prevented AF accumulation by A. flavus CA28 and reduced AF accumulation 10-fold by A. flavus CA43. No decrease in AF accumulation was apparent when TX9-8 was inoculated 24 h after toxigenic L- or S strain A. flavus isolates started growing so the competitive effect likely is due to TX9-8 outgrowing toxigenic A. flavus isolates .
In 2009, it was reported that Serratia plymuthica 5-6, isolatedfromthe rhizosphere of pea reduced dry rot of potato caused by Fusarium sambucinum . In 2009, a new strain of Bacillus pumilus isolated from Korean soybean sauce showed strong antifungal activity against the AF-producing fungi A. flavus and A.parasiticus .
In 2010, a strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated by Kong and collaborators for its activity in reducing postharvest decay of peanut kernels caused by Aspergillus flavus in in vitro and in vivo tests, this, because microorganisms are capable of producing many unique bioactive substances, and therefore could be a rich resource for antagonists . The results showed that the concentrations of antagonist had a significant effect on biocontrol effectiveness in vivo: when the concentration of the washed bacteria cell suspension was used at 1×109 CFU/ml, the percentage rate of rot of peanut kernels was 31.67%±2.89%, which was markedly lower than that treated with water (the control) after 7 days of incubation at 28 °C. The results also showed that unwashed cell culture of B. megaterium was as effective as the washed cell suspension, and better biocontrol was obtained when longer incubation time of B. megaterium was applied. When the incubation time of B. megaterium was 60 h, the rate of decay declined to 41.67%±2.89%. Furthermore, relative to the expression of 18S rRNA, the mRNA abundances of aflR gene and aflS gene in the experiment group were 0.28±0.03 and 0.024±0.005 respectively, indicating that this strain of B. megaterium could significantly reduce the biosynthesis of AF and expression of aflR gene and aflS gene .
In 2011, Degola and collaborators conducted a study in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing AF accumulation when grown in mixed cultures together with atoxigenic strains; additionally, they developed a simple and inexpensive procedure that might be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co- infection .
Farzaneh and collaborators reported in this year, an investigation in which Bacillus subtilis strain UTBSP1 was isolated from pistachio nuts and studied for the degradation of AFB1. The results indicated B. subtilis UTBSP1 could considerably remediate AFB1 from nutrient broth culture and pistachio nut by 85.66% and 95%, respectively. Cell free supernatant fluid caused an apparent 78.39% decrease in AFB1 content. The optimal conditions for AFB1 degradation by cell free supernatant appeared at 35 and 40°C, during 24 h. Furthermore, the results indicated that AFB1 degradation is enzymatic and responsible enzymes are extracellular and constitutively produced. They found that destructive AFB1 differed from standard AFB1 chemically, and lost a fluorescence property .
It was found that A. flavus K49 produces neither AFs nor cyclopiazonic acid (CPA) and is currently being tested in corn-growing fields in Mississippi. Its lack of production of AF and CPA results from single nucleotide mutations in the polyketide synthase gene and hybrid polyketide nonribosomal peptide synthase gene, respectively. Furthermore, based on single nucleotide polymorphisms of the AF biosynthesis omtA gene and the CPA biosynthesis dmaT gene, it is known that K49, AF36 and TX9-8 form a biocontrol group, appear to be derived from recombinants of typical large and small sclerotial morphotype strains .
Not only Aspergillus, but also other pathogens have been faced to biocontrol. For example, it is known that the plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, it was performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). Genes that encode proteins with potential role in biological control have been tested. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani .
It is a fact that several papers have been published about AFB1 reduction by some bacterial isolates. Lactic acid bacteria such as Lactobacillus, Bifidobacterium, Propionibacterium and Lactococcus were found to be active in removing AFB1 primarily by the adhesion method. In addition, some bacteria such as Rhodococcus erythropolis, Bacillus sp., Stenotrophomonas maltophilia, Mycobacterium fluoranthenivorans and Nocardia corynebacterioides were reported to degrade AFB1 .
5.2.2. Natural products and essential oils
Plants produce lots of secondary metabolites as part of their normal growth and development in order to fight against environmental stress, pathogen attack or other adversities. One of the most important secondary metabolites are essential oils (EOs), which are extracted from plants, commonly by a distillation process  and then used as natural additives in different foods to reduce the proliferation of microorganisms and their toxins production due to their antifungal, antiviral, antibacterial, antioxidant and anticarcinogenic properties [70-72]. They have received major consideration in regard to their relatively safe status and enrichment by a wide range of structurally different useful constituents . Until 1989, more than 1340 plants were known to be potential sources of antimicrobial compounds, which are safe for the environment and consumers, and are useful to control postharvest diseases, being an excellent alternative to reduce the use of synthetic chemicals in agriculture. The majorities of the essential oils are classified as Generally Recognized As Safe (GRAS) and have low risk for developing resistance to pathogenic microorganisms [74, 75].
There is a large number of different groups of chemical compounds present in EOs, that is why antimicrobial activity is not attributable to one specific mechanism but to the existence of several targets in the cell [76, 77]. There is a relationship between the chemical structures of the most abundant compounds in the EOs and the anitimicrobial activity; minor components have a critical part to play in antimicrobial activity, possibly by producing a synergic effect between other components . Not only EOs but also alkaloids, phenols, glycosides, steroids, coumarins and tannins have been found to have antimicrobial properties . Generally, the extent of the inhibition of the oils could be attributed to the presence of an aromatic nucleus containing a polar functional group , being phenols the majority group. For example, in 2008, Bluma and Etcheverry, based in the principle that phenolics are secondary metabolites synthesized via phenylpropanoid biosynthetic pathway which build blocks for cell wall structures serving as defense against pathogens, found that phenolic compounds such as acetocyringone, syringaldehyde and sinapinic acid inhibit AFB1 biosynthesis by A. flavus in PDA and reduce norsolinic acid production, because the presence of phenolic OH groups are able to form hydrogen bonds with the active sites of target enzymes increasing antimicrobial activity .
There is a wide list of natural products from the entire world (summarized in Table 1) used in the last decade to diminish Aspergillus populations to counteract the effect of AFs in food or to test fumigant activity in feed at specific inhibitory concentrations . It has been demonstrated that the antifungal capability of those EOs depend on the concentration in which they are applied and the conditions around them. In 2001, Varma and Dubey reported that EOs from plants like Caesulia axillaris and Mentha arvensis have fumigant activity in the management of biodeterioration of stored wheat samples by A. flavus showing the same efficacy as postharvest fungicides used for this purpose . In 2002, Soliman and Badeaa tested inhibitory activity of essential oils from 12 medicinal plants against A. flavus, A. parasiticus, A. ochraceus and Fusarium moniliforme, finding that the oils of thyme and cinnamon (at a 4500 ppm concentration), marigold (42000 ppm), spearmint, basil and quyssum (3000 ppm) completely inhibit all the test fungi. Caraway was inhibitory at 2000 ppm against A. flavus, A. parasiticus and 3000 ppm against A. ochraceaus and F. moniliforme. A. flavus, A. ochraceus, A. parasiticus and F. moniliforme were completely inhibited by anise at 4500 ppm, being chamomile and hazanbul essential oils just partially effective against the test toxigenic fungi .
|NATURAL PRODUCT||COMMON NAME||PRINCIPAL METABOLITE||PATHOGEN INHIBITED||INHIBITORY CONCENTRATION||REFERENCE|
|Achillea fragrantissima||Qyssum||Polyphenolic compounds||A. flavus,|
|Agave asperrima||Maguey Cenizo||Polyphenolic compounds||A. flavus|
|< 2 mg ml-1|||
|Agave striata||Maguey Espadín||Polyphenolic compounds||A. flavus|
|< 2 mg ml-1|||
|Ageratum conyzoides||Goatweed||Precocene, Cumarine, trans-Caryophyllene||A.flavus||0.10 µg ml-1|||
|Azadirachta indica A. Juss||Neem||Aromatic compounds||A. parasiticus||"/ 10% (v/v)|||
|Caesulia axillaris||Pink Node Flower||Aromatic compounds||A. flavus||nd|||
|Calendula ofricinalis L.||Marigold||Carfone||A. flavus,|
|< 2,000 ppm|||
|Carum carvi L.||Caraway||Carfone||A. flavus,|
|2,000 – 3,000 ppm|||
|Cicuta virosa L. var. latisecta Celak||Umbelliferae||γ-Terpinene p-Cymene Cumin Aldehyde||A. flavus||5 µl ml-1|||
|Cinnamomum cassia||Cassia||Aromatic compounds||A. parasiticus||2.5 % (v/v)|||
|Cinnamomum zeylanicum L.||Cinnamon||Cinnamic aldehyde|
|200 – 250 ppm,|
< 500 ppm
|[71, 83, 85]|
|Citrus limon||Lemon||Limomene||A. flavus||2, 000 ppm|||
|A.flavus||1 – 5%,|
|Eucalyptus globulus||Blue Gum||1,8-cineole||A.flavus|
|2,000 – 3,000 µg g-1|||
|Bay Leaf||Aromatic compounds||A. parasiticus||1 – 5 % (v/v)|||
|Lippia turbinate var. integrifolia (griseb)||Poleo||β-Cariofilene|
|2,000 – 3,000 µg g-1,|
2500 μl l -1
|Mentha arvensis||Wild Mint||Menthone|
|Ocimum basilicum||Sweet Basil||β-pinene α-pinene|
|A. parasiticus||5% (v/v)||[71, 79]|
|Ocimum basilicum L||Basil||β-pinene α-pinene|
|Ocimum gratissimum||Clove Basil||γ-terpinene|
|800 ppm||[83, 93]|
|A. flavus||500 µg g-1,|
100 – 2,000 ppm
|2,000 – 3,000 µg g-1,|
2500 μl l -1
|Pimpinella anisum L.||Anise||Metilchavicol Anethol||A. flavus,|
|< 500 ppm|||
|Satureja hortensis L.||Winter Savory||Carvacrol Thymol||A. parasiticus||~0.5 mM||[81, 87]|
|1500 μl l -1|||
|Thymus eriocalyx||Avishan||Thymol β-phellandrene cis-sabinene hydroxide 1,8-cineole β-pinene||A. parasiticus||250 ppm|||
|Thymus vulgaris L.||Thyme||β-pinene α-pinene|
Thymol p- cymene
|< 500 ppm,|
|Thymus X-porlock||Thyme||Thymol β-phellandrene cis-sabinene hydroxide 1,8-cineole β-pinene||A. parasiticus||250 ppm|||
|Trachyspermum ammi (L.)||Ajowan||Aromatic compounds||A. flavus||1 g ml-1|||
|Zingiber officinale||Ginger||Polyphenolic compounds||A.flavus||800 – 2,500 ppm|||
Metabolites obtained from some natural products which are used to diminish fungal populations and AF production (nd= no data).
EOs and other natural products have been tested not only against Aspergillus species but also Fusarium species, which most of the times are developed in parallel. In 2003, Vellutti and collaborators reported the effect of cinnamon, clove, oregano, palmarose and lemongrass oils on fumonisin B1 growth and production by three different isolates of F. proliferatum in irradiated maize grain at 0.995 and 0.950 aw and at 20 and 30°C. The five essential oils inhibited growth of F. proliferatum isolates at 0.995 aw at both temperatures, while at 0.950 aw only cinnamon, clove and oregano oils were effective in inhibiting growth of F. proliferatum at 20°C and none of them at 30°C. Cinnamon, oregano and palmarose oils had significant inhibitory effect on FB1 production by the three strains of F. proliferatum at 0.995 aw and both temperatures, while clove and lemongrass oils had only significant inhibitory effect at 30°C . In 2004, Nguefack and his group of researchers tested the inhibitory effect of EOs extracted from Cymbopogon citratus, Monodora myristica, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale against F. moniliforme, being O. gratissimum, T. vulgaris and C. citratus the most effective over conidial germination and fungal growth at 800, 1000 and 1200 ppm, respectively. Moderate activity was observed for the EO from Z. officinale between 800 and 2500 ppm, while the EO from M. myristica was less inhibitory. These effects against food spoilage and mycotoxin producing fungi indicated the possible ability of each EO as a food preservative .
In 2005, Sánchez and collaborators prepared ethanolic, methanolic and aqueous extracts of flowers from mexican Agave asperrima and Agave striata, in order to diminish growth and production of AF from A. flavus and A. parasiticus at in vitro and in vivo level. All extracts, but specifically the methanolic one, showed an effective inhibition growth (99%) . In the same year, Rasooli & Owlia extracted the EOS from Thymus eriocalyx and Thymus X-porlock in order to test antifungal activity against A. parasiticus growth and AF production. T. eriocalyx showed lethal effects at 250 ppm while T. X-porlock was lethal at 500 ppm .
EOs from common spices have been also investigated, that is the case of cinnamon (Cinnamomum zeylanicum) and oregano (Origanum vulgare) which shows antifungal activity against A. flavus at 2000 ppm and 1000 ppm respectively in a malt-agar medium and a fungistatic activity at 100 ppm. . Eucalyptus (Eucalyptus globules) is effective against the storage fungi A. flavus and A. parasiticus . Lemon EO (Citrus limon), applied in food AF-contaminated samples, results in a strong antiaflatoxigenic and antifungal substance, reducing AF concentrations in food samples for broilers up to 73.6% . Sweet basil (Ocimum basilicum), cassia (Cinnamomum cassia), coriander (Coriandrum sativum) and bay leaf (Laurus nobilis) at 1–5% (v/v) concentration were studied in palm kernel over the aflatoxigenic fungus A. parasiticus CFR 223 and AF production. Sweet basil oil at optimal protective dosage of 5% (v/v) was fungistatic on A. parasiticus; in contrast, oils of cassia and bay leaf stimulated the mycelia growth of the fungus in vitro but reduced the AF concentration (AFB1+AFG1) of the fungus by 97.92% and 55.21% respectively, while coriander oil did not have any effect on both the mycelia growth and AF content of the fungus. The combination of cassia and sweet basil oils at half their optimal protective dosages (2.5% v/v) completely inhibited the growth of the fungus. It was found that the addition of whole and ground basil leaves markedly reduced AF contamination; however, 10% (w/w) of whole leaves was more effective as the reduction in AF was between 89.05% and 91% .
In 2008, Bluma and Etcheverry found that Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) had an inhibitory effect on Aspergillus section Flavi growth rate, and their efficacy depended mainly on the water activity and EOs concentration. Boldus, poleo, and mountain thyme EOs completely inhibited AFB1 at 2000 and 3000 µg g-1 . Satureja hortensis L. has been also reported as a potent inhibitor of AFB1 and AFG1 produced by A. parasiticus at concentrations from 0.041 to 1.32 mM . In 2009, Kumar and collaborators found that Cymbopogon flexuosus EO and its components were efficient in checking fungal growth and AF production, inhibiting absolutely inhibited the growth of A. flavus and AFB1 production at 1.3 µlml-1 and 1.0 µlml-1 respectively, due to the principal component: eugenol . Razzaghi-Abyaneh and his investigation group found that Thymus vulgari and Citrus aurantifolia inhibit both A. parasiticus and AF production. The EOs from Mentha spicata L., Foeniculum miller, Azadirachta indica A. Juss, Conium maculatum and Artemisia dracunculus only inhibited fungal growth, while Carum carvi L. effectively inhibited AF production without any obvious effect on fungal growth. Ferula gummosa, Citrus sinensis, Mentha longifolia and Eucalyptus camaldulensis had no effect on A. parasiticus growth and AF production at all concentrations used . There are other investigations of the potential use of antifungal component eugenol for the reduction of AFB1. Komala and collaborators reported some findings in stored sorghum grain due to fungal infestation of sorghum results in deterioration of varied biochemical composition of the grain. In this study, three genotypes (M35-1; C-43; LPJ) were inoculated with two highly toxigenic strains of Aspergillus flavus with three different eugenol treatments in order to evaluate the AFB1 production. From this study it was found that at 8.025 mg/g concentration, eugenol completely inhibited the AFB1 production. The lowest amount of AFB1 was observed in genotype M35-1, whereas higher amount AFB1 was observed in LPJ followed by C-43. In all sorghum genotypes there was a significant positive correlation existing between protein content and AF produced, the r values being 0.789 and 0.653, respectively. Starch in three genotypes was found to have a significant negative correlation with AF produced. The starch content decreased whereas the protein content in all sorghum varieties increased during infection .
Ageratum conyzoides EO is other specie that has been studied recently. It acts directly on the mycelial growth and AFB1 production by A. flavus, inhibiting fungal growth to different extents depending on the concentration, and completely inhibiting AF production at concentrations above 0.10 µg/mL, because this EO acts affecting mainly the fungal mitochondria . This EO acts similarly than Ajowan extract (Trachyspermum ammi L., which acts directly over AFB1, AFB2 and AFG2 . In 2011, it was found that Ocimum gratissimum EO acts a nontoxic antimicrobial and antiaflatoxigenic agent against fungal and AF contamination of spices infected with A. flavus isolated from Piper nigrum and Myristica fragrans respectively at 0.6 μl/ml and 0.5 μl/ml, as well as a shelf life enhancer in view of its antioxidant activity, playing a prominent role in the development of an ideal plant based food additive . It was found too that EOs extracted from the fruits of Cicuta virosa L. var. latisecta Celak acts against A. flavus, A, oryzae, A. niger, and Alternaria alternata, having a strong inhibitory effect on spore production and germination in all tested fungi proportional to concentration. The oil exhibited noticeable inhibition on dry mycelium weight and synthesis of AFB1 by A. flavus, completely inhibiting AFB1 production at 4 μL/mL .
Because of the great results obtained with this kind of AFs biocontrol, researchers are still investigating new natural products and their active compounds in order to deal with those toxins ad the fungi which produce them, and avoiding the use of fumigants that are toxic for plants and for plant consumers. In this year, EOs from plants like Zanthoxylum alatum Roxb have been studied, because it has been proved that its two major constituents (linalool and methyl cinnamate) inhibit the growth of a toxigenic strain of A. flavus (LHP-10) as well as AFB1 secretion at different concentrations. Zanthoxylum alatum Roxb EO has also showed strong antioxidant activity with an IC50 value at 5.6 μl/ml . EOs from boldo, clove, anise and thyme are still studied against aflatoxigenic Aspergillus strains in specific cultures like peanut-based medium, finding that those EOs have influence on lag phase, growth rate, and AFB1 accumulation . The EO extracted from the bark of Cinnamomum jensenianum Hand. Mazz has been tested for antifungal activity against A. flavus. Mycelial growth and spore germination was inhibited by the oil in a dose-dependent manner. The oil also exhibited a noticeable inhibition on the dry mycelium weight and the synthesis of AFB1 by A. flavus, completely restraining AFB1 production at 6 µl/ml. The possible mode of action of the oil against A. flavus is discussed based on changes in the mycelial ultrastructure . Nevertheless, most research is needed in order to understand the mechanisms of action of the essential oils over aflatoxigenic fungi, turning them into potential sources for food preservation.