Vibrio gazogenes inhibits aflatoxin production through downregulation of aflatoxin biosynthetic genes in Aspergillus flavus

Aspergillus flavus is an opportunistic pathogen of oilseed crops such as maize, peanut, cottonseed, and tree nuts and produces carcinogenic secondary metabolites known as aflatoxins during seed colonization. Aflatoxin contamination not only reduces the value of the produce but also is a health hazard to humans and animals. Previously, we observed inhibition of A. flavus aflatoxin biosynthesis upon exposure to the marine bacterium, Vibrio gazogenes (Vg). In this study, we used RNA sequencing to examine the transcriptional profiles of A. flavus treated with both live and heat-inactivated dead Vg and control samples. Fungal biomass, total accumulated aflatoxins, and expression profiles of genes constituting secondary metabolite biosynthetic gene clusters were determined at 24, 30, and 40 h after treatment. Statistically significant reductions in total aflatoxins were detected in Vg-treated samples as compared to control samples at 40 h. But no statistical difference in fungal biomass was observed upon these treatments. The Vg treatments were most effective on aflatoxin biosynthesis as was reflected in significant downregulation of majority of the genes in the aflatoxin gene cluster including the aflatoxin pathway regulator gene, aflR. Along with aflatoxin genes, we also observed significant downregulation in some other secondary metabolite gene clusters including cyclopiazonic acid and aflavarin, suggesting that the treatment may inhibit other secondary metabolites as well. Finally, a weighted gene correlation network analysis identified an upregulation of ten genes that were most strongly associated with Vg-dependent aflatoxin inhibition and provide a novel start-point in understanding the mechanisms that result in this phenomenon.

Spatial and temporal population dynamics of Aspergillus flavus in commercial pistachio orchards in Arizona

Aspergillus flavus infects a wide range of crops, including pistachios, and subsequent aflatoxin contamination results in significant economic losses. Application of biocontrol products based on non-aflatoxigenic (atoxigenic) strains of A. flavus is one of the most effective tactics for controlling aflatoxins in crops. Both risk of aflatoxin contamination and effectiveness of biocontrol are influenced by the extent to which A. flavus spores move into pistachio tree canopies during periods of nut development. Thus, the purpose of this study was to evaluate spatial and temporal population dynamics of A. flavus, including the applied biocontrol strain AF36, in canopies of pistachio orchards in Arizona. Propagule densities of A. flavus were quantified on leaf samples collected from lower, middle, and upper canopies from spring through harvest in 2018 and 2019. Aspergillus flavus propagule densities peaked during periods of high temperature and rainfall in 2018 (up to 600 CFU/g) and 2019 (up to 23 CFU/g), which coincided with nut development and maturation. The applied biocontrol strain AF36 was detected at all canopy heights, but overall propagule densities were greater in the upper and middle canopy (mean = 70 CFU/g) compared to the lower canopy (mean = 47 CFU/g). Results suggest June to August is the period during which A. flavus inoculum increases in Arizona pistachio orchards, and to most effectively displace aflatoxin-producing fungi in tree canopies, biocontrol applications should precede this period. In addition, this study demonstrates that soil-applied biocontrol strains can successfully disperse throughout the canopies of commercial tree nut orchards.

Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers.

Ozone to Overcome Aspergillus flavus and Aflatoxin in Grains: Opportunities and Challenges of Implementation

<p>Ozone can be used as an alternative to control mold and aflatoxins in grains that is more eco-friendly because it does not leave residues that are harmful for humans, animals and environment. The use of ozone was quite effective in reducing mold and aflatoxin in grains such as barley, whole wheat, corn and rice. In Indonesia, ozone was limited used for sterilization of fruit and vegetable. Therefore, the comprehensive review on the potential of ozone in grains is needed, especially on the priority commodities of agricultural development in Indonesia, such as rice and corn. The objective of this review was to examine the opportunities of ozone to reduce Aspergillus flavus and aflatoxin in grains, so that it can improve its quality and shelf life. Many studies showed that the use of ozone reduced Aspergillus flavus and aflatoxin in grains by 50-90%.<br />Keywords: Grains, Aspergillus flavus, aflatoxin, ozone</p><p> </p><p><strong>Abstrak</strong></p><p><strong>OZON UNTUK MENGATASI CEMARAN ASPERGILLUS FLAVUS DAN</strong><br /><strong>AFLATOKSIN PADA BIJI-BIJIAN: PELUANG DAN TANTANGAN</strong><br /><strong>IMPLEMENTASI</strong></p><p>Ozon dapat digunakan sebagai salah satu alternatif pengendalian cendawan dan aflatoksin pada biji-bijian yang lebih ramah lingkungan karena tidak meninggalkan residu yang berbahaya bagi manusia, hewan, maupun lingkungan. Penggunaan ozon cukup efektif mengurangi kontaminasi cendawan dan aflatoksin pada bijibijian seperti barley, biji gandum, jagung, dan beras. Di Indonesia, ozon digunakan secara terbatas untuk proses pencucian beberapa jenis buah dan sayuran. Oleh karena itu diperlukan telaah lebih lanjut mengenai potensi penggunaan ozon pada biji-bijian terutama komoditas strategis yang menjadi prioritas dalam pembangunan pertanian di Indonesia seperti padi dan jagung. Tujuan dari tulisan ini adalah untuk menelaah peluang penggunaan ozon dalam mengurangi kontaminasi Aspergillus flavus dan cemaran aflatoksin pada produk biji-bijian, sehingga diharapkan dapat memperbaiki kualitas dan meningkatkan umur simpan produk. Hasil penelitian menunjukkan penggunaan ozon dapat menurunkan cemaran A. flavus dan aflatoksin pada biji-bijian sampai 50-90%.<br />Kata kunci: Biji-bijian, Aspergillus flavus, aflatoksin, ozon</p>