Research

17 pages, 2964 KiB

Open AccessArticle

Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in Aspergillus parasiticus

by Masayuki Ichinomiya, Ayaka Kawamoto, Takahiro Yamaguchi, Keiko Iwashita, Hitoshi Nagashima, Hidemi Hatabayashi, Hiromitsu Nakajima and Kimiko Yabe

J. Fungi 2023, 9(1), 51; https://doi.org/10.3390/jof9010051 - 29 Dec 2022

Cited by 1 | Viewed by 1672

Abstract

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that [...] Read more.

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that unlike CTN, the CTN-KA adduct does not show antimicrobial activity against Escherichia coli or Bacillus subtilis or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. To examine the function of KA production by fungi, we isolated A. parasiticus mutants with impaired KA production. When the mutants were incubated in either liquid or agar medium supplemented with CTN, they were more susceptible to CTN than the wild KA-producing strain. The same results were obtained when we used the A. oryzae KA-producing strain RIB40 and KA-non-producing strains. When KA was added to the CTN-containing agar medium, the inhibition of growth by CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN’s toxicity. We also observed that CTN enhanced the production of KA by A. parasiticus as well as A. oryzae strains. Reverse transcription-PCR showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. However, the enhancement of KA production with CTN was repressed by the addition of α-tocopherol or butylated hydroxy anisole, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. In contrast, α−tocopherol did not affect inhibition of AF production as well as fungal growth by CTN, suggesting that the regulation of these inhibitions with CTN might be different from that of KA production. We propose a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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18 pages, 4522 KiB

Open AccessEditor’s ChoiceArticle

Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct

by Masayuki Ichinomiya, Emi Fukushima-Sakuno, Ayaka Kawamoto, Hiroyuki Nakagawa, Hidemi Hatabayashi, Hiromitsu Nakajima and Kimiko Yabe

J. Fungi 2023, 9(1), 29; https://doi.org/10.3390/jof9010029 - 23 Dec 2022

Cited by 2 | Viewed by 2686

Abstract

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not [...] Read more.

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB₂·AFG₂. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named “CTN-KA adduct”. Our examination of the metabolites’ toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN’s toxicity was alleviated with KA by converting CTN to the CTN-KA adduct. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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14 pages, 4101 KiB

Open AccessArticle

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing

by Haruhisa Suga, Masahiro Hayashi, Masayo Kushiro, Norichika Miyano, Hiroyoshi Inoue, Kaori Nakajima, Taku Kawakami, Takuji Tonooka, Takashi Nakajima, Masafumi Shimizu and Koji Kageyama

J. Fungi 2022, 8(10), 1048; https://doi.org/10.3390/jof8101048 - 05 Oct 2022

Cited by 1 | Viewed by 1814

Abstract

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used [...] Read more.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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18 pages, 3651 KiB

Open AccessArticle

Antifungal Potential of Azotobacter salinestris Strain Azt 31 against Phytopathogenic Fusarium spp. Associated with Cereals

by Hanumanthu Nagaraja, Gurikar Chennappa, Nagaraj Deepa, Manjunath Krishnappa Naik, Kadaiah Ajithkumar, Yatgal Sharanappa Amaresh, Premila N. Achar and M. Y. Sreenivasa

J. Fungi 2022, 8(5), 473; https://doi.org/10.3390/jof8050473 - 30 Apr 2022

Cited by 2 | Viewed by 2179

Abstract

Antifungal efficacy of Azotobacter salinestris against trichothecene-producing Fusarium spp. was investigated in maize, sorghum, and wheat. The three cereals were subjected to four treatments as control (T1), Fusarium alone (T2), combination of Fusarium and A. salinestris treatment (T3), and only A. salinestris (T4). [...] Read more.

Antifungal efficacy of Azotobacter salinestris against trichothecene-producing Fusarium spp. was investigated in maize, sorghum, and wheat. The three cereals were subjected to four treatments as control (T1), Fusarium alone (T2), combination of Fusarium and A. salinestris treatment (T3), and only A. salinestris (T4). All the treatments were evaluated for total mass of seedlings, root and shoot length, seed germination, and vigor index (VI), and extent of rhizoplane colonization by A. salinestris was investigated. Further, greenhouse studies were conducted to learn the efficacy of A. salinestris in vivo conditions. Antifungal efficacy was tested by the dual-culture method which resulted in significant reduction in Fusarium growth. Infection by Fusarium was reduced up to 50% in treated cereals such as maize, sorghum, and wheat, and there was also significant increase in seedling mass in the three hosts. Maize showed the highest VI (1859.715), followed by sorghum (1470.84), and wheat (2804.123) with A. salinestris treatment. In addition, seed germination was enhanced to 76% in maize, 69% in sorghum, and 68% in wheat, respectively. Efficacy of rhizoplane colonization showed successful isolation of A. salinestris with high CFU rate, and furthermore, significant colonization inhibition by Fusarium spp. was observed. In the greenhouse conditions, on the 45th day of the experimental set-up, the highest shoot length/root length recorded in maize was 155.70/70.0 cm, in sorghum 165.90/48.0 cm, and in wheat 77.85/56.0 cm, and the maximum root mass recorded was 17.53 g in maize, 4.52 g in sorghum, and 1.90 g in wheat. Our present study showed that seed treatment by A. salinestris, may be used as an alternate biocontrol method against Fusarium infection in maize, sorghum, and wheat. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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16 pages, 19431 KiB

Open AccessArticle

Characterization of Fusarium acuminatum: A Potential Enniatins Producer in Tunisian Wheat

by Yasmine Chakroun, Souheib Oueslati, Laetitia Pinson-Gadais, Manef Abderrabba and Jean-Michel Savoie

J. Fungi 2022, 8(5), 458; https://doi.org/10.3390/jof8050458 - 28 Apr 2022

Cited by 1 | Viewed by 2228

Abstract

Fusarium Head Blight (FHB), caused by multiple species of Fusarium in small grain cereals, is a significant and long-standing problem anywhere in the world. Knowing regional Fusarium spp. present on non-symptomatic grains and their potential for mycotoxin production is of concern for identifying [...] Read more.

Fusarium Head Blight (FHB), caused by multiple species of Fusarium in small grain cereals, is a significant and long-standing problem anywhere in the world. Knowing regional Fusarium spp. present on non-symptomatic grains and their potential for mycotoxin production is of concern for identifying novel actions for FHB and mycotoxin management, such as treatments with essential oils. Analyzing the mycotoxin content of grains from non-symptomatic ears of different wheat varieties cultivated in Tunisia, we isolated Fusaria specimens identified as F. culmorum and F. acuminatum using analysis of the partial DNA sequence of the β-tubulin gene and ITS region. Two isolates of the latter species, uncommon in cereal grains in this region until now, were shown to be effective producers of enniatins in vitro, with 1390 and 3089 µg g⁻¹ mycelial biomass (dry) in 11-day-old cultures. The susceptibility of an isolate of F. acuminatum to the fungistatic and antimycotoxin effects of eight essential oils was measured. Essential oils from Ammoides pusilla and Thymus capitatus used at 0.1 µL mL⁻¹ in an agar culture medium, affected the mycelial growth by 55% and 79%, respectively and reduced the accumulation of enniatins per unit of mycelial colony by 26% and 52%, respectively. Finally, F. acuminatum was shown to be a contaminant of wheat grains in Tunisia and it may contribute to the contamination in enniatins. Two essential oils of Tunisian plants could be used for developing a biofungicide limiting both its mycelial growth and its accumulation of mycotoxins in grains. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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13 pages, 832 KiB

Open AccessArticle

Occurrence and Risk Assessment of Dietary Exposure to Deoxynivalenol in Wheat-Based Products Based Different Wheat-Producing Area for the Inhabitants in Shanghai, China

by Xianli Yang, Zhiyong Zhao, Jianhua Wang, Junhua Yang, Hengchao E, Bo Chen, Pengzhen He, Yanglan Tan and Changyan Zhou

J. Fungi 2021, 7(12), 1015; https://doi.org/10.3390/jof7121015 - 26 Nov 2021

Cited by 3 | Viewed by 1783

Abstract

Deoxynivalenol (DON) is one of the major mycotoxins that contaminate cereals. In this study, we determined the DON level in wheat-based products from Chinese five main production areas collected in Shanghai and calculated the daily intake of DON for inhabitants using the point [...] Read more.

Deoxynivalenol (DON) is one of the major mycotoxins that contaminate cereals. In this study, we determined the DON level in wheat-based products from Chinese five main production areas collected in Shanghai and calculated the daily intake of DON for inhabitants using the point evaluation and the probabilistic evaluation based on Monte Carlo simulation. The results showed the positive rates of DON in the products were higher than 80.0%, with the concentrations ranging from 41.8 to 1110 µg/kg. The estimated mean daily intakes of DON for 7- to 10-year-old children and adults groups were below 1 µg/kg bw/day, the provisional maximum tolerable daily intake (PMTDI) set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), suggesting no health risks for the consumers. However, the 99th percentiles of dietary DON exposures for children and adults exceeded the PMTDI, indicating adverse health effects might occur if the two groups intake highly contaminated wheat-based products. The potential health risks for the two groups exposed to DON in the wheat-based products from the Middle and Lower Yangtze Valley (MLYV) were higher than those from the other areas in China. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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22 pages, 4736 KiB

Open AccessArticle

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins

by Gang Liu, Dong-Yun Zuo, Peng Yang, Wei-Jie He, Zheng Yang, Jing-Bo Zhang, Ai-Bo Wu, Shu-Yuan Yi, He-Ping Li, Tao Huang and Yu-Cai Liao

J. Fungi 2021, 7(11), 941; https://doi.org/10.3390/jof7110941 - 06 Nov 2021

Cited by 1 | Viewed by 1910

Abstract

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this [...] Read more.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, ₆₇HXXXG₇₁, and exhibits DON affinity with a dissociation constant (K_D) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC₅₀ values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium–wheat interactions. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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16 pages, 3643 KiB

Open AccessArticle

Analysis of Stored Wheat Grain-Associated Microbiota Reveals Biocontrol Activity among Microorganisms against Mycotoxigenic Fungi

by Manoj Kumar Solanki, Ahmed Abdelfattah, Sudharsan Sadhasivam, Varda Zakin, Michael Wisniewski, Samir Droby and Edward Sionov

J. Fungi 2021, 7(9), 781; https://doi.org/10.3390/jof7090781 - 20 Sep 2021

Cited by 6 | Viewed by 3508

Abstract

Wheat grains are colonized by complex microbial communities that have the potential to affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities have been shown to protect plants against pathogens through antagonism. We evaluated the role of [...] Read more.

Wheat grains are colonized by complex microbial communities that have the potential to affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities have been shown to protect plants against pathogens through antagonism. We evaluated the role of the microbiome in seed health: in particular, against mycotoxin-producing fungi. Amplicon sequencing was used to characterize the seed microbiome and determine if epiphytes and endophytes differ in their fungal and bacterial diversity and community composition. We then isolated culturable fungal and bacterial species and evaluated their antagonistic activity against mycotoxigenic fungi. The most prevalent taxa were found to be shared between the epiphytic and endophytic microbiota of stored wheat seeds. Among the isolated bacteria, Bacillus strains exhibited strong antagonistic properties against fungal pathogens with noteworthy fungal load reduction in wheat grain samples of up to a 3.59 log₁₀ CFU/g compared to untreated controls. We also found that a strain of the yeast, Rhodotorula glutinis, isolated from wheat grains, degrades and/or metabolizes aflatoxin B₁, one of the most dangerous mycotoxins that negatively affects physiological processes in animals and humans. The mycotoxin level in grain samples was significantly reduced up to 65% in the presence of the yeast strain, compared to the untreated control. Our study demonstrates that stored wheat grains are a rich source of bacterial and yeast antagonists with strong inhibitory and biodegradation potential against mycotoxigenic fungi and the mycotoxins they produce, respectively. Utilization of these antagonistic microorganisms may help reduce fungal and mycotoxin contamination, and potentially replace traditionally used synthetic chemicals. Full article

(This article belongs to the Special Issue Mycotoxins and Toxigenic Fungi)

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