Mycotoxigenic fungal growth inhibition and multi-mycotoxin reduction of potential biological control agents indigenous to grain maize.

The present work investigated the potential of fungal species from grain maize farms in Malaysia as antagonists against the indigenous mycotoxigenic fungal species and their subsequent mycotoxin production. Dual-culture assay was conducted on grain maize agar (GMA) with 12 strains of potential fungal antagonists namely Bjerkandra adusta, Penicillium janthinellum, Schizophyllum commune, Trametes cubensis, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma harzianum, and Trichoderma yunnanense against seven mycotoxigenic strains namely Aspergillus flavus, Aspergillus niger, Fusarium verticillioides, and Fusarium proliferatum producing aflatoxins, ochratoxin A, and fumonisins, respectively. Based on fungal growth inhibition, Trichoderma spp. showed the highest inhibitory activity (73-100% PIRG, Percentage Inhibition of Radial Growth; 28/0 ID, Index of Dominance) against the tested mycotoxigenic strains. Besides, B. adusta and Tra. cubensis showed inhibitory activity against some of the tested mycotoxigenic strains. All fungal antagonists showed varying degrees of mycotoxin reduction. Aflatoxin B1 produced by A. flavus was mainly reduced by P. janthinellum, Tra. cubensis, and B. adusta to 0 ng/g. Ochratoxin A produced by A. niger was mainly reduced by Tri. harzianum and Tri. asperellum to 0 ng/g. Fumonisin B1 and FB2 produced by F. verticillioides was mainly reduced by Tri. harzianum, Tri. asperelloides, and Tri. asperellum to 59.4 and 0 µg/g, respectively. Fumonisin B1 and FB2 produced by F. proliferatum were mainly reduced by Tri. asperelloides and Tri. harzianum to 244.2 and 0 µg/g, respectively. This is the first study that reports on the efficacy of Tri. asperelloides against FB1, FB2, and OTA, P. janthinellum against AFB1, and Tra. cubensis against AFB1.

Diversity of endophytic fungi isolated from different plant parts of Acacia mangium, and antagonistic activity against Ceratocystis fimbriata, a causal agent of Ceratocystis wilt disease of A. mangium in Malaysia.

Acacia mangium is an important wood for commercial products especially pulp and medium-density fibreboard. However, it is susceptible to Ceratocystis fimbriata infection, leading to Ceratocystis wilt. Therefore, the present work aimed to (i) establish the diversity of endophytic fungi in different plant parts of A. mangium,and (ii) evaluate the antifungal potentials of the isolated and identified endophytic fungi against C. fimbriata. Endophytic fungal identification was conducted by PCR amplification and sequencing of the internal transcribed spacer 1 (ITS1) and ITS4 regions of nuclear ribosomal DNA. A total of 66 endophytic fungi were successfully isolated from different parts of A. mangium; leaf (21), stem (13), petiole (12), root (9), flower (6), and fruit (5). The endophytic fungal isolates belonged to Ascomycota (95.5%) and Zygomycota (4.5%). For Ascomycota 13 genera were identified: Trichoderma (28.6%), Nigrospora (28.6%), Pestalotiopsis (12.7%), Lasiodiplodia (9.5%), Aspergillus (6.3%), Sordariomycetes (3%), and Neopestalotiopsis, Pseudopestalotiopsis, Eutiarosporella, Curvularia, Fusarium, Penicillium, and Hypoxylon each with a single isolate. For Zygomycota, only Blakeslea sp. (5%) was isolated. Against C. fimbriata, Trichoderma koningiopsis (AC 1S) from stem, Nigrospora oryzae (AC 7L) from leaf, Nigrospora sphaerica (AC 3F) from the flower, Lasiodiplodia sp. (AC 2 U) from fruit, Nigrospora sphaerica (AC 4P) from petiole, and Trichoderma sp. (AC 9R) from root exhibited strong inhibition for C. fimbriata between 58.33 to 69.23%. Thus, it can be concluded that certain endophytic fungi of A. mangium have the potential to be harnessed as anti-Ceratocystis agent in future biotechnological applications.

Antagonism of nonaflatoxigenic Aspergillus flavus isolated from peanuts against aflatoxigenic A. flavus growth and aflatoxin B1 production in vitro.

Aspergillus section Flavi constitutes several species of opportunistic fungi, notable among them are A. flavus and A. parasiticus, capable of surviving harsh conditions and colonizing a wide range of agricultural products pre- and postharvest. Physical and chemical control methods are widely applied in order to mitigate the invasion of A. flavus in crops. However, physical control is not suitable for large scale and chemical control often leads to environmental pollution, whereas biological control offers a safer, environmentally friendly, and economical alternative. The present study aimed to investigate the antagonism of several non-aflatoxigenic A. flavus strains against the aflatoxigenic ones in vitro (semisynthetic peanut growth medium; MPA) in terms of colony growth rate and AFB1 inhibition. Different peanut concentrations were used to obtain the optimum peanut concentration in the formulated growth medium. A dual culture assay was performed to assess the antagonism of nonaflatoxigenic strains against the aflatoxigenic ones. Results revealed that 9% MPA exhibited the highest growth and AFB1 inhibition by nonaflatoxigenic strains. It was also found that different nonaflatoxigenic strains exhibited different antagonism against the aflatoxigenic ones which ranged from 11.09 ± 0.65% to 14.06 ± 0.14% for growth inhibition, and 53.97 ± 2.46% to 72.64 ± 4.54% for AFB1 inhibition. This variability could be due to the difference in antagonistic metabolites produced by different nonaflatoxigenic strains assessed in the present study. Metabolomics study to ascertain the specific metabolites that conferred the growth and aflatoxin inhibition is ongoing.

Assessment of multi-mycotoxin contamination throughout the supply chain of maize-based poultry feed from selected regions of Malaysia by LC-MS/MS.

The objective of this study was to examine the occurrence of multi-mycotoxin contamination throughout the supply chain of maize-based poultry feed. Different sampling points throughout the feed supply chain were selected from two companies that manufactured the poultry feed. A total of 51 samples, consisting of grain maize and maize-based poultry feeds, were collected. The samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the occurrence of multi-mycotoxin. The results revealed that 100% of samples throughout the maize-based poultry feed supply chain were spoiled with more than one mycotoxin. Fumonisin B1 (8.02-1,220 µg/kg) and fumonisin B2 (11.1-1,109 µg/kg) were the main mycotoxins detected at all sampling points throughout the feed supply chain. Zearalenone (ZEA) (6.63-7.50 µg/kg) was also detected in 11.7% (n = 6) (out of a total of 51) samples. As the supply chain progresses, a reduction in mycotoxin contamination was observed. Aflatoxins, ochratoxin A (OTA), deoxynivalenol (DON), HT-2, and T-2 toxin were not detected. The levels of mycotoxins detected throughout the supply chain were below the international regulatory limits, thus indicating the low risk of exposure to mycotoxins in maize-based poultry feed in Malaysia. Nevertheless, due to the presence of multiple ingredients in most food and feed, efforts to understand and address challenges associated with mycotoxins throughout the entire supply chain need to be more holistic to protect public health.

Aflatoxin Biosynthesis, Genetic Regulation, Toxicity, and Control Strategies: A Review.

Aflatoxins (AFs) are highly toxic and cancer-causing compounds, predominantly synthesized by the Aspergillus species. AFs biosynthesis is a lengthy process that requires as minimum as 30 genes grouped inside 75 kilobytes (kB) of gene clusters, which are regulated by specific transcription factors, including aflR, aflS, and some general transcription factors. This paper summarizes the status of research on characterizing structural and regulatory genes associated with AF production and their roles in aflatoxigenic fungi, particularly Aspergillus flavus and A. parasiticus, and enhances the current understanding of AFs that adversely affect humans and animals with a great emphasis on toxicity and preventive methods.

Biocontrol of Aflatoxins Using Non-Aflatoxigenic Aspergillus flavus: A Literature Review.

Aflatoxins (AFs) are mycotoxins, predominantly produced by Aspergillus flavus, A. parasiticus, A. nomius, and A. pseudotamarii. AFs are carcinogenic compounds causing liver cancer in humans and animals. Physical and biological factors significantly affect AF production during the pre-and post-harvest time. Several methodologies have been developed to control AF contamination, yet; they are usually expensive and unfriendly to the environment. Consequently, interest in using 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. AF- strains cannot produce AFs due to the absence of polyketide synthase genes or genetic mutation. AF- strains competitively exclude the AF+ strains in the field, giving an extra advantage to the stored grains. Several microbiological, molecular, and field-based approaches have been used to select a suitable biocontrol agent. The effectiveness of biocontrol agents in controlling AF contamination could reach up to 99.3%. Optimal inoculum rate and a perfect time of application are critical factors influencing the efficacy of biocontrol agents.

Climatic conditions and farm practices affected the prevalence of Aspergillus section Flavi on different types of dairy goat's feed.

The present work aimed to determine the prevalence of aflatoxigenic Aspergillus section Flavi on different types of dairy goat's feed samples obtained from four dairy goat's farms around the central region of Peninsular Malaysia, and to examine the effects of climatic conditions (temperature, relative humidity) of the dairy goat's farms, and their feeding and storage practices on the fungal prevalence of different types of dairy goat's feed. A total of 60 goat's feed samples were obtained, and their proximate composition and water activity were determined, following which they were cultivated on DRBC and AFPA for total fungal load and Aspergillus section Flavi load determination, respectively. Fungal isolates were identified morphologically, and toxigenicity potentials of Aspergillus section Flavi isolates were determined using CCA. The temperature and relative humidity data of all farms were obtained from the Malaysian Meteorological Department. The total fungal loads (on DRBC) of the goat's feed samples were log 0.767 to 7.071 CFU/g which included the common feed contaminants such as Aspergillus, Fusarium, and Penicillium. The Aspergillus section Flavi loads (on AFPA) were log 0.667 to 3.206 CFU/g. Farm A yielded the highest number of Aspergillus section Flavi isolates as well as the highest number of aflatoxigenic isolates. It was found that climatic conditions and different practices between farms positively influenced the fungal prevalence on goat's feed samples based on the Pearson correlation analysis. The prevalence of mycotoxigenic isolates on goat's feed warrants for urgent intervention to ensure that goats are being fed with nutritionally adequate and safe feed. The presence of aflatoxigenic Aspergillus section Flavi isolates indicates the risk of aflatoxin B1 contamination on the goat's feed, aflatoxicosis development in the goats, and aflatoxin M1 bio-transformation in the goat's milk. This is a potential threat to the flourishing goat's milk industry in Malaysia.

Phytopathogenic organisms and mycotoxigenic fungi: Why do we control one and neglect the other? A biological control perspective in Malaysia.

In this review, we present the current information on development and applications of biological control against phytopathogenic organisms as well as mycotoxigenic fungi in Malaysia as part of the integrated pest management (IPM) programs in a collective effort to achieve food security. Although the biological control of phytopathogenic organisms of economically important crops is well established and widely practiced in Malaysia with considerable success, the same cannot be said for mycotoxigenic fungi. This is surprising because the year round hot and humid Malaysian tropical climate is very conducive for the colonization of mycotoxigenic fungi and the potential contamination with mycotoxins. This suggests that less focus has been made on the control of mycotoxigenic species in the genera Aspergillus, Fusarium, and Penicillium in Malaysia, despite the food security and health implications of exposure to the mycotoxins produced by these species. At present, there is limited research in Malaysia related to biological control of the key mycotoxins, especially aflatoxins, Fusarium-related mycotoxins, and ochratoxin A, in key food and feed chains. The expected threats of climate change, its impacts on both plant physiology and the proliferation of mycotoxigenic fungi, and the contamination of food and feed commodities with mycotoxins, including the discovery of masked mycotoxins, will pose significant new global challenges that will impact on mycotoxin management strategies in food and feed crops worldwide. Future research, especially in Malaysia, should urgently focus on these challenges to develop IPM strategies that include biological control for minimizing mycotoxins in economically important food and feed chains for the benefit of ensuring food safety and food security under climate change scenarios.

Aspergillus section Flavi and Aflatoxins: Occurrence, Detection, and Identification in Raw Peanuts and Peanut-Based Products Along the Supply Chain.

Aflatoxin contamination in foods is a global concern as they are carcinogenic, teratogenic and mutagenic compounds. The aflatoxin-producing fungi, mainly from the Aspergillus section Flavi, are ubiquitous in nature and readily contaminate various food commodities, thereby affecting human's health. The incidence of aflatoxigenic Aspergillus spp. and aflatoxins in various types of food, especially raw peanuts and peanut-based products along the supply chain has been a concern particularly in countries having tropical and sub-tropical climate, including Malaysia. These climatic conditions naturally support the growth of Aspergillus section Flavi, especially A. flavus, particularly when raw peanuts and peanut-based products are stored under inappropriate conditions. Peanut supply chain generally consists of several major stakeholders which include the producers, collectors, exporters, importers, manufacturers, retailers and finally, the consumers. A thorough examination of the processes along the supply chain reveals that Aspergillus section Flavi and aflatoxins could occur at any step along the chain, from farm to table. Thus, this review aims to give an overview on the prevalence of Aspergillus section Flavi and the occurrence of aflatoxins in raw peanuts and peanut-based products, the impact of aflatoxins on global trade, and aflatoxin management in peanuts with a special focus on peanut supply chain in Malaysia. Furthermore, aflatoxin detection and quantification methods as well as the identification of Aspergillus section Flavi are also reviewed herein. This review could help to shed light to the researchers, peanut stakeholders and consumers on the risk of aflatoxin contamination in peanuts along the supply chain.

Molecular Characterisation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus Section Flavi Isolated from Imported Peanuts along the Supply Chain in Malaysia.

Peanuts are widely consumed in many local dishes in southeast Asian countries, especially in Malaysia which is one of the major peanut-importing countries in this region. Therefore, Aspergillus spp. and aflatoxin contamination in peanuts during storage are becoming major concerns due to the tropical weather in this region that favours the growth of aflatoxigenic fungi. The present study thus aimed to molecularly identify and characterise the Aspergillus section Flavi isolated from imported peanuts in Malaysia. The internal transcribed spacer (ITS) and ß-tubulin sequences were used to confirm the species and determine the phylogenetic relationship among the isolates, while aflatoxin biosynthesis genes (aflR, aflP (omtA), aflD (nor-1), aflM (ver-1), and pksA) were targeted in a multiplex PCR to determine the toxigenic potential. A total of 76 and one isolates were confirmed as A. flavus and A. tamarii, respectively. The Maximum Likelihood (ML) phylogenetic tree resolved the species into two different clades in which all A. flavus (both aflatoxigenic and non-aflatoxigenic) were grouped in the same clade and A. tamarii was grouped in a different clade. The aflatoxin biosynthesis genes were detected in all aflatoxigenic A. flavus while the non-aflatoxigenic A. flavus failed to amplify at least one of the genes. The results indicated that both aflatoxigenic and non-aflatoxigenic A. flavus could survive in imported peanuts and, thus, appropriate storage conditions preferably with low temperature should be considered to avoid the re-emergence of aflatoxigenic A. flavus and the subsequent aflatoxin production in peanuts during storage.

The effects of different packaging materials, temperatures and water activities to control aflatoxin B1 production by Aspergillus flavus and A. parasiticus in stored peanuts.

Aflatoxins (AFs) are secondary metabolites produced by aflatoxigenic strains of Aspergillus flavus and A. parasiticus, the most toxic being aflatoxin B1 (AFB1). The purpose of the present work was to investigate the effects of industrial-grade packaging materials (low-density polyethylene, polypropylene, polyethylene-laminated aluminium); temperatures (25 °C, 30 °C); and water activities (0.74 a w, 0.85 a w) on AFB1 production by A. flavus and A. parasiticus in stored peanut kernels. Commercially-obtained samples were segregated into packaging materials, separately inoculated with the aflatoxigenic Aspergillus spp., and stored for 1 month under various °C + a w regimes. AFB1 production was quantified by high performance liquid chromatography with fluorescence detector (HPLC-FLD). For A. flavus in PELA, no AFB1 was detected (100% reduction) at 25 °C for both a w tested. For A. parasiticus in PELA, no AFB1 was detected at 25 °C (0.85 a w) and 30 °C (0.74 a w). Highest concentration of AFB1 was detected in LDPE for both A. flavus (46.41 ppb) and A. parasiticus (414.42 ppb), followed by PP (A. flavus 24.29 ppb; A. parasiticus 386.73 ppb). In conclusion, storing peanut kernels in PELA in a dry place at room temperature has been demonstrated as an adequate and inexpensive method in inhibiting growth of Aspergillus spp. and lowering AFB1 contamination in peanuts.

Formulation of maize- and peanut-based semi-synthetic growth media for the ecophysiological studies of aflatoxigenic Aspergillus flavus in maize and peanut agro-ecosystems.

In studying the ecophysiology of fungal phytopathogens, several stages are involved (in vitro, greenhouse, in planta). Most in vitro studies extensively utilise the general growth media such as Potato Dextrose Agar and Malt Extract Agar. Although the crop components in these media serve as excellent carbon sources and yield luxuriant growth, they are not naturally contaminated with Aspergillus flavus and thus might result in under- or overestimation of its actual toxigenic potentials. Empirical data on the formulation of semi-synthetic growth medium mimicking the natural crop commonly contaminated by A. flavus for the ecophysiological studies in vitro are scarce. The present work was aimed at investigating the ecophysiology of A. flavus on commercial growth media (PDA, MEA); formulating maize- and peanut-based semi-synthetic growth media using two methods of raw material preparation (milling, hot water extraction) at different concentrations (1, 3, 5, 7, 9% w/v), and comparing the ecophysiological parameters between commercial and formulated growth media. Growth rates were obtained by computing the hyphal expansion data into y = mx + c equation. AFB1 was quantified using high performance liquid chromatography with fluorescence detector. Formulated media were found to yield significantly higher growth rates when compared to commercial media. However, commercial media yielded significantly higher AFB1 when compared to all formulated media. Between the two formulations, milling yielded significantly higher growth rates and AFB1 when compared to hot water extraction. Although in vitro data cannot directly extrapolate in planta performance, results obtained in the present work can be used to gauge the actual toxigenic potential of A. flavus in maize and peanut agro-ecosystems.

Efficacy of fungal and bacterial antagonists for controlling growth, FUM1 gene expression and fumonisin B1 production by Fusarium verticillioides on maize cobs of different ripening stages.

This study was carried out to examine the efficacy of two biocontrol agents (Clonostachys rosea 016, BCA1; Gram-negative bacterium, BCA5) for control of FUM1 gene expression and fumonisin B1 (FB1) production by F. verticillioides FV1 on maize cobs of different ripening stages: R3, Milk (0.985 aw); R4, Dough (0.976 aw); R5, Dent (0.958 aw). Initially, temporal studies on FUM1 gene expression and FB1 production were performed on maize kernels for up to 14days. This revealed that day 10 was optimum for both parameters, and was used in the biocontrol studies. Maize cobs were inoculated with 50:50 mixtures of the pathogen:antagonist inoculum and incubated in environmental chambers to maintain the natural aw conditions for ten days at 25 and 30°C. The growth rates of F. verticillioides FV1, the relative expression of the FUM1 gene and FB1 production were quantified. It was found that, aw×temp had significant impacts on growth, FUM1 gene expression and FB1 production by F. verticillioides FV1 on maize cobs of different maturities. The fungal antagonist (BCA1) significantly reduced FB1 contamination on maize cobs by >70% at 25°C, and almost 60% at 30°C regardless of maize ripening stage. For the bacterial antagonist (BCA5) however, FB1 levels on maize cobs were significantly decreased only in some treatments. These results suggest that efficacy of antagonists to control mycotoxin production in ripening maize cobs needs to take account of the ecophysiology of the pathogen and the antagonists, as well as the physiological status of the maize during silking to ensure effective control.

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia.

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4 × 10(4) to 2.1 × 10(6) CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100%), followed by Penicillium chrysogenum (62%), Aspergillus niger (54%), and Aspergillus flavus (44%). Citrinin was present in 100% samples (0.23-20.65 mg/kg), aflatoxin in 92% samples (0.61-77.33 µg/kg) and Ochratoxin-A in 100% samples (0.23-2.48 µg/kg); 100% citrinin and 76.09% aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.