Non-Aflatoxigenic Aspergillus flavus Control Aflatoxins
Aflatoxins (AFs) are carcinogenic compounds causing liver cancer in humans and animals. Several methodologies have been developed to control AF contamination, yet; they are usually expensive and unfriendly to the environment. Consequently, interest in biocontrol agents has increased, as they are convenient, advanced, and friendly to the environment. Using non-aflatoxigenic strains of A. flavus (AF−) as biocontrol agents is the most promising method to control AFs’ contamination in cereal crops.
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Aflatoxins (AFs) are secondary metabolites produced by Aspergillus flavus, A. parasiticus, A. nomius, and A. pseudotamarii [1,2]. AFs are organic compounds with lower molecular weight, typically produced by fungal mycelia and accumulated in conidia and sclerotia. AFs contaminate various crops, including corn, oilseeds, rice, and nuts [3,4,5,6]. AFs contamination in cereals may occur during pre- or post-harvest stages [7,8]. Hot temperature and high humidity stimulate fungal growth in fields and storage. Contamination by AFs is responsible for substantial commercial losses throughout the world [9,10,11]. AFs are among the most toxic compounds that adversely affect humans and animals’ health [12,13,14,15,16,17]. AFs are mutagenic, teratogenic, genotoxic, and carcinogenic compounds, causing severe diseases in humans, poultry, fishes, and cattle under long-term exposure [18,19]. AFs can penetrate the feed and food chain, posing a threat to even newborns [20,21]. While several AFs were currently identified, AFB1, AFB2, AFG1, and AFG2 are the four most significant AFs. The IARC (International Agency for Research on Cancer) classifies AFB1 as the most toxic, mutagenic, and Group 1 human carcinogen [22,23,24], causing chronic and acute diseases in children and the elderly. AFB1 carcinogenicity has long been linked to the liver; however, recent epidemiological studies revealed that it was also carcinogenic to the pancreas, kidney, bone, bladder, and central nervous system [25,26,27,28].
2. Advantages of Biocontrol of Aflatoxins Using Non-Aflatoxigenic Aspergillus flavus
Biocontrol methods are more effective and innovative in controlling AF contamination in crops. The application of biocontrol agents (AF−) carries some adaptations in fungal populations, which persist throughout the food chain. These adaptations prevent the grains from AF contamination during storage and transport; even environmental conditions favor fungal growth. In biocontrol methods, AF− strain application in the field remarkably reduces AF contamination in crops [29,30]. Similarly, like air, AF can disperse Aspergillus spores-communities, improve safety within the treated, and positively affect neighboring fields . The positive impacts of AF− strains can benefit crops and other plants for several years. This means a single dose of AF− strain could benefit the treated crop and the second season crop, which missed the treatment .
3. Selection of Non-Aflatoxigenic Strains
Biocontrol is a promising method to reduce AF contamination in crops. Recent studies reported reducing AF contamination by applying AF− strain to the soil around growing plants. When the crop is vulnerable to fungal attack during drought conditions, these AF− strains competitively exclude the AF+ strains in the soil and reduce AF concentrations. Dorner  reported the reduction in AF contamination in a cornfield using AF− strains. In other research, Dorner  assessed the efficacy of AF− for AFs control in peanuts. AF− strains can be found in air, soil, and plants. Usually, both AF+ and AF− strains mutually occur in different ecosystems. AF− strains competing with AF+ strains for nutrients provide an opportunity to use them as biocontrol agents. Different techniques have been developed to discover the suitable AF− strain for biocontrol use. Some of them are based on phylogenetic features, while others on phenotypic characteristics such as sclerotial size. Based on sclerotial morphology and production, A. flavus can be divided into two distinct morphotypes: S-strain and L-strain. The S-strains produce a large number of small-sized sclerotia (>400 µm in diameter), whereas the L-strains produce a small number of large-sized sclerotia (<400 µm in diameter). Moreover, S-strains produce a higher concentration of AF compared to L-strains. Molecular techniques may describe the phylogenetic relationships between A. flavus strains successfully. Several polymerase chain reaction (PCR)-based pyrosequencing methods are currently being developed to detect genes responsible for AF production and discover suitable biocontrol agents . Abbas et al.  isolated some AF− strains, including K49, F3W4, NRRL 58,974, NRRL 58,976, and NRRL 58,988. The classification was based on their growth rate, pigmentation, fluorescence, and AF production.
4. Efficacy of Non-Aflatoxigenic Strains as Biocontrol Agents
AF− strains have been suggested as biocontrol agents hoping that they would inhibit the growth of AF+ and thereby reduce AFs contamination. Previous studies conducted by Erhlich  revealed that co-inoculation of AF− strains with AF+ substantially reduced the production of AF in corn under in vitro conditions. The potential for biocontrol of AFs using AF− strains has been demonstrated under field conditions in cotton , peanuts , and corn [39,40]. These scientists have applied the AF− strain to the soil as infested grain cultures of barley, rice, or wheat, whereas  inoculated corn ears directly by injection. In the cotton studies performed by Cotty , the AF− strains failed to suppress AFs contamination when sprayed on the cottonseed immediately before the bolls formed but were effective when sprayed on the soil later.
5. Factors Affecting the Efficacy of Biocontrol Agents
5.1. Inoculation Method
For many years, AF− strains have been used on cornfield soil. Although the use of K49 in the soil can reduce AF levels by 65% , the direct use of AF− strain on corn ears is immensely more efficient. A clay-based water-dispersible granule system was also developed to spray AF− strain on corn silk directly. This management decreased AF production by up to 97%.
5.2. Inoculum Rate
Inoculum concentration is an essential factor for the effective control of AF contamination. Recent studies have revealed a direct relationship between the inoculum rate and AF’s efficacy− strain decreasing AF concentrations . Studies demonstrated a significant reduction in AF concentration in peanuts when AF− inoculum increased from 2–50 g/L. In the USA, research was conducted in which an AF− strain (NRRL 21,368) with different quantities (0, 2, 10, and 50 g) was applied to the cornfield .
5.3. Optimal Time for Non-Aflatoxigenic Strains Application
Research showed that with the concentration of AF− strains, the time of its application significantly affects their efficacy. The application of AF− strain at earlier stages significantly reduced AF levels in cotton. Similarly, Kabak and Dobson  suggested the co-inoculation of AF+ and AF− strains (TX9-8) to reduce the AF contamination; however, if the AF− strain is applied one day later, AF+ strains, fewer or no reduction in AF concentrations will be achieved.
5.4. Abiotic Factors
The time for the application of AF−, depends on the significant environmental conditions. Abiotic factors such as water activity and temperature directly affect AF− strains’ efficacy by controlling spore germination, hyphal growth, and spore-production .
5.5. Biotic Factors
Low temperature and high water content in storage provide favorable conditions for insects, mites, and other microorganisms to grow. Insects’ respiration process produces hot spots in seeds, causing grain charring that affects seed quality and germination. In grains, insects’ activities increase the surrounding bulk’s temperature and water content, providing favorable mold growth conditions. Studies have shown that seeds damaged by insects are highly susceptible to fungal contamination . Some fungi absorb insects and boost their populace, while others repel pests by secreting harmful toxins.
5.6. Physiological Manipulation of Non-Aflatoxigenic Strains
Most fungal niches are not persistent as they modify their features according to the external environment . In unfavorable environments, xerophilic fungi produce small polyols, which allow their enzymatic systems to work efficiently. Similarly, A. flavus accumulates glycerol and erythritol in their conidia during unfavorable conditions . Therefore, fungal propagules used for biocontrol must be resistant to environmental stresses . According to Magan , agricultural management could improve the resistive performance of biocontrol agents.
- Frisvad, J.C.; Hubka, V.; Ezekiel, C.N.; Hong, S.B.; Nováková, A.; Chen, A.J.; Arzanlou, M.; Larsen, T.O.; Sklenár, F.; Mahakarnchanakul, W.; et al. Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins, and other mycotoxins. Stud. Mycol. 2019, 93, 1–63.
- Pankaj, S.; Shi, H.; Keener, K.M. A review of novel physical and chemical decontamination technologies for aflatoxin in food. Trends Food Sci. Technol. 2017, 71, 73–83.
- Lizárraga-Paulín, E.G.; Miranda-Castro, S.P.; Moreno-Martínez, E.; Torres-Pacheco, I.; Lara-Sagahón, A.V. Novel methods for preventing and controlling aflatoxins in food: A worldwide daily challenge. In Aflatoxins-Recent Advances and Future Prospects; Razzaghi-Abyaneh, M., Ed.; InTech: Rijeka, Croatia, 2013; pp. 93–128.
- Burger, H.; Shephard, G.; Louw, W.; Rheeder, J.; Gelderblom, W. The mycotoxin distribution in maize milling fractions under experimental conditions. Int. J. Food Microbiol. 2013, 165, 57–64.
- Lombard, M.J. Mycotoxin exposure and infant and young child growth in Africa: What do we know? Ann. Nutr. Metab. 2014, 64 (Suppl. 2), 42–52.
- Wagacha, J.; Muthomi, J. Mycotoxin problem in Africa: Current-status, implications for food safety and health and possible management strategies. Int. J. Food Microbiol. 2008, 124, 1–12.
- Iimura, K.; Furukawa, T.; Yamamoto, T.; Negishi, L.; Suzuki, M.; Sakuda, S. The mode of action of cyclo (L-Ala-L-Pro) in inhibiting aflatoxin production of Aspergillus flavus. Toxins 2017, 9, 219.
- Shuaib, F.M.; Ehiri, J.; Abdullahi, A.; Williams, J.H.; Jolly, P.E. Reproductive health effects of aflatoxins: A review of the literature. Reprod. Toxicol. 2010, 29, 262–270.
- Bhat, R.; Rai, R.V.; Karim, A.A. Mycotoxins in food and feed: Present status and future concerns. Compr. Rev. Food Sci. Food Saf. 2010, 9, 57–81.
- Chen, M.T.; Hsu, Y.H.; Wang, T.S.; Chien, S.W. Mycotoxin monitoring for commercial foodstuffs in Taiwan. J. Food Drug Anal. 2016, 24, 147–156.
- Williams, J.H.; Phillips, T.D.; Jolly, P.E.; Stiles, J.K.; Jolly, C.M.; Aggarwal, D. Human aflatoxicosis in developing countries: A review of toxicology, exposure, potential health consequences, and interventions. Am. J. Clin. Nutr. 2004, 80, 1106–1122.
- Fan, S.; Li, Q.; Sun, L.; Du, Y.; Xia, J.; Zhang, Y. Simultaneous determination of aflatoxin B1 and M1 in milk, fresh milk, and milk powder by LC-MS/MS utilizing online turbulent flow chromatography. Food Addit. Contam. A, 2015, 32, 1175–1184.
- Giovati, L.; Magliani, W.; Ciociola, T.; Santinoli, C.; Conti, S.; Polonelli, L. AFM1 in milk: Physical, biological, and prophylactic methods to mitigate contamination. Toxins 2015, 7, 4330–4349.
- Monson, M.S.; Cardona, C.J.; Coulombe, R.A.; Reed, K.M. Hepatic transcriptome responses of domesticated and wild turkey embryos to aflatoxin B1. Toxins, 2016, 8, 16.
- Rajkovic, A.; Uyttendaele, M.; Debevere, J. Computer-aided boar semen motility analysis for cereulide detection in different food matrices. Int. J. Food Microbiol. 2007, 114, 92–99.
- Verma, R.; Nair, A. Ameliorative effect of vitamin E on aflatoxin-induced lipid peroxidation in the testis of mice. Asian J. Androl. 2001, 3, 217–221.
- Yuan, S.; Wu, B.; Yu, Z.; Fang, J.; Liang, N.; Zhou, M.; Peng, X. The mitochondrial and endoplasmic reticulum pathways involved in the apoptosis of bursa of Fabricius cells in broilers exposed to dietary aflatoxin B1. Oncotarget, 2016, 7, 65295.
- Peles, F.; Sipos, P.; Gyori, Z.; Pfliegler, W.P.; Giacometti, F.; Serraino, A.; Pagliuca, G.; Gazzotti, T.; Pócsi, I. Adverse effects, transformation, and channeling of aflatoxins into food raw materials in livestock. Front. Microbiol. 2019, 10, 286.
- Jalili, M. A review on aflatoxins reduction in food. Iran. J. Health Saf. Environ. 2015, 3, 445–459.
- Maleki, F.; Abdi, S.; Davodian, E.; Haghani, K.; Bakhtiyari, S. Exposure of infants to aflatoxin M1 from mother’s breast milk in Ilam, Western Iran. Osong Public Health Res. Perspect. 2015, 6, 283–287.
- Warth, B.; Braun, D.; Ezekiel, C.N.; Turner, P.C.; Degen, G.H.; Marko, D. Biomonitoring of mycotoxins in human breast milk: Current state and future perspectives. Chem. Res. Toxicol. 2016, 29, 1087–1097.
- Ali, N.; Hashim, N.H.; Shuib, N.S. Natural occurrence of aflatoxins and ochratoxin A in processed spices marketed in Malaysia. Food Addit. Contam. Part- A, 2015, 32, 518–532.
- McGuire, S. World cancer report, 2014. Geneva, Switzerland: World Health Organization, the international agency for cancer research, WHO Press, 2015. Adv. Nutr. 2016, 7, 418–419.
- Fouad, M.A.; Ruan, D.; El-Senousey, K.H.; Chen, W.; Jiang, S.; Zheng, C. Harmful effects and control strategies of aflatoxin B1 produced by Aspergillus flavus and Aspergillus parasiticus strains on poultry: Review. Toxins (Basel) 2019, 11, 176.
- Benkerroum, N. Mycotoxins in dairy products: A review. Int. Dairy J. 2016, 62, 63–75.
- McGlynn, K.A.; London, W.T. Epidemiology and natural history of hepatocellular carcinoma. Best Pract. Res. Clin. Gastroenterol. 2005, 19, 3–23.
- Male, D. Challenges Facing Foodborne Mycotoxin Regulation and Public Health Consequences of Exposure in Children under 5 Years of Age. Ph.D. Thesis, Michigan State University, East Lansing, MI, USA, 2017; pp. 1–166.
- El-Serag, H.B. Epidemiology of hepatocellular carcinoma. Liver Biol. Pathol. 2020, 758–772.
- Chulze, S. Strategies to reduce mycotoxin levels in maize during storage: A review. Food Addit. Contam. 2010, 27, 651–657.
- Rani, P.R.; Chelladurai, V.; Jayas, D.S.; White, N.D.G.; Kavitha-Abirami, C.V. Storage studies on pinto beans under different moisture contents and temperature regimes. J. Stored Prod. Res. 2013, 52, 78–85.
- Probst, C.; Bandyopadhyay, R.; Cotty, P.J. Diversity of aflatoxin-producing fungi and their impact on food safety in sub-Saharan Africa. Int. J. Food Microbiol. 2014, 174, 113–122.
- Bandyopadhyay, R.; Kumar, M.; Leslie, J.F. Relative severity of aflatoxin contamination of cereal crops in West Africa. Food Addit. Contam. 2007, 24, 1109–1114.
- Donner, M.; Atehnkeng, J.; Sikora, R.A.; Bandyopadhyay, R.; Cotty, P.J. Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria. Food Addit. Contam. 2010, 27, 576–590.
- Das, D.; Selvaraj, R.; Bhat, M.R. Optimization of inulinase production by a newly isolated strain Aspergillus flavus var. flavus by solid-state fermentation of Saccharum arundinaceum. Biocatal. Agric. Biotechnol. 2019, 22, 101363.
- Abbas, H.K.; Zablotowicz, R.M.; Weaver, M.A.; Shier, W.T.; Bruns, H.A.; Bellaloui, N.; Abel, C.A. Implications of Bt traits on mycotoxin contamination in maize: Overview and recent experimental results in the Southern United States. J. Agric. Food Chem. 2013, 61, 11759–11770.
- Ehrlich, K. Effect on aflatoxin production of competition between wild type and mutant strains of Aspergillus parasiticus. Mycopathologia, 1987, 97, 93–96.
- Cotty, P.J. Aflatoxin-producing potential of communities of Aspergillus section Flavi from cotton-producing areas in the United States. Mycol. Res. 1997, 101, 698–704.
- Dorner, J.W.; Cole, R.J.; Connick, W.J.; Daigle, D.L.; McGuire, M.R.; Shasha, B.S. Evaluation of biological control formulations to reduce aflatoxin contamination in peanuts. Biol. Control. 2003, 26, 318–324.
- Brown, R.I.; Cotty, P.J.; Cleveland, T.E. Reduction in aflatoxin content of maize by atoxigenic strains of Aspergillus flavus. Food Prot. 1991, 54, 623–626.
- Cotty, P.J. Effect of harvest date on aflatoxin contamination of cottonseed. Plant. Dis. 1991, 75, 312–314.
- Brown, R.L.; Chen, Z.Y.; Cleveland, T.E.; Russin, J.S. Advances in the development of host resistance in corn to aflatoxin contamination Aspergillus flavus. Phytopathology 1999, 89, 113–117.
- Cotty, P.J. Influence of field application of an atoxigenic strain of Aspergillus flavus on the populations of A. flavus infecting cotton bolls and the aflatoxin content cottonseed. Phytopathology 1994, 84, 1270–1277.
- Accinelli, C.; Mencarelli, M.; Saccà, M.L.; Vicari, A.; Abbas, H.K. Managing and monitoring Aspergillus flavus in corn using bioplastic-based formulations. Crop. Prot. 2012, 32, 30–35.
- Lewis, M.H.; Carbone, I.; Luis, J.M.; Payne, G.A.; Bowen, K.L.; Hagan, A.K.; Kemerait, R.; Heiniger, R.; Ojiambo, P.S. Biocontrol strains differentially shift the genetic structure of indigenous soil populations of Aspergillus flavus. Front. Microbiol. 2019, 10, 1738.
- Mamo, F.T.; Selvaraj, J.N.; Wang, Y.; Liu, Y. Recent Developments in the Screening of Atoxigenic Aspergillus flavus towards Aflatoxin Biocontrol. J. Appl. Environ. Microbiol. 2017, 5, 20–30.
- Kabak, B.; Dobson, A.D. Biological strategies to counteract the effects of mycotoxins. J. Food Prot. 2009, 72, 2006–2016.
- Köhl, J.; Langerak, C.J.; Meekes, E.T.M.; Molhoek, W.M.L. Biological control of Alternaria radicina in seed production of carrots with Ulocladium atrum. Seed Sci. Tech. 2004, 32, 857–861.
- Reddy, K.R.N.; Reddy, C.S.; Muralidharan, K. Potential of botanicals and biocontrol agents on growth and aflatoxin production Aspergillus flavus infecting rice grains. Food Control. 2009, 20, 173–178.
- Magan, N. Ecophysiology of biocontrol agents for improved competence in the phyllosphere. Microb. Ecol. Aerial Plant. Surf. 2006, 1, 149–164.
- Gasch, A.P. Comparative genomics of the environmental stress response in ascomycete fungi. Yeast, 2007, 24, 961–976.
- Köhl, J.; Molhoek, W. Effect of water potential on conidial germination and antagonism of Ulocladium atrum against Botrytis cinerea. Phytopathology 2001, 91, 485–491.
This entry is adapted from 10.3390/jof7050381