Tenuazonic acid production is dispensable for virulence, but its biosynthetic gene expression pattern is associated with the infection of Pyricularia oryzae

Tenuazonic acid (TeA) is a toxin produced by the rice blast fungus Pyricularia oryzae. Although knockout of the TeA biosynthetic gene TAS1 did not affect the virulence of P. oryzae, constitutive TAS1 expression suppressed its infection. TAS1 expression was induced alongside transition of P. oryzae infection behavior. The results suggested that controlling TeA biosynthesis is important for P. oryzae infection.

Contamination of grain in West Siberia by Alternaria fungi and their mycotoxins

The ubiquitous occurrence of Alternaria fungi belonging to sections Alternaria and Infectoriae was confirmed using real-time PCR in wheat, barley and oat grain grown in West Siberia in 2018‒2019. The DNA amount of Alternaria section Alternaria fungi varied from 53×10-4 to 21731×10-4 pg/ng and on average exceeded the DNA amount of Alternaria section Infectoriae fungi by 4.5‒14.6 times, depending on the crop and harvest year.The average DNA amount of Alternaria fungi belonging to both sections in the oat grain was lower than in wheat and barley grain. The grain samples from Altay region were the most infected with Alternaria fungi. The alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA) mycotoxins produced by Alternaria fungi were detected by HPLC-MS/MS in 23 %, 6 %, 85 %, and 83 % of analyzed grain samples, respectively. The majority (61 %) of the samples contained two Alternaria mycotoxins in the grain (mainly TEN and TeA), 19 % of the samples three mycotoxins, and only one sample all four together. In the most of samples the content of Alternaria mycotoxins did not exceed 100 μg/kg, and only TeA content was higher (from 113 to 14963 μg/kg) than others. The significant differences in grain crops by the Alternaria mycotoxins content were revealed: more amounts of AOH, AME, and less amount of TEN were found in oat grain then in barley grain. A high positive significant correlation between the DNA amount of Alternaria section Alternaria fungi and TeA was established that indicates the role of these fungi as the main producers of TeA in the grain.

Evaluation of prophylactic efficacy of cinnamaldehyde in murine model against Paradendryphiella arenariae mycotoxin tenuazonic acid-induced oxidative stress and organ toxicity

Cinnamaldehyde (Cin) is a natural product obtained from cinnamon and is reported to have a potential anti-fungal, anti-oxidant, anti-inflammatory and anticancer effect. The present study investigated the possible protective role of Cin against tenuazonic acid-induced mycotoxicity in the murine model. Tenuazonic acid (TeA), a toxin produced by Alternaria is a common contaminant in tomato and tomato-based products. Here, Swiss male mice were administered with TeA isolated from Paradendryphiella arenariae (MW504999) (source-tomato) through injection (238 µg/kg BW) and ingestion (475 µg/kg BW) routes for 2 weeks. Thereafter, the prophylaxis groups were treated with Cin (210 mg/kg BW). The experiment was carried out for 8 weeks. The treated groups were compared to the oral and intra-peritoneal experimental groups that received the toxin solely for 8 weeks. Haematological, histopathological and biochemical aspects of the experimental and the control mice were analysed. Sub-chronic intoxication of mice with TeA showed elevated malondialdehyde (MDA), reduced catalase (CAT) and superoxide dismutase (SOD) production; abnormal levels of aspartate transaminase (AST) and alanine transaminase (ALT). Treatment with Cin reversed TeA-induced alterations of antioxidant defense enzyme activities and significantly prevented TeA-induced organ damage. Thus, cinnamaldehyde showed therapeutic effects and toxicity reduction in TeA induced mycotoxicosis.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid

Study of fungal antibiotics in their competitive interactions with arthropods may lead to development novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained by various methods showed a wide range of biological activity, including entomotoxic properties. Analysis of their composition and bioactivity allowed to reveal several known mycotoxins and unidentified compounds that may be involved in entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with LT50 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited growth of G. mellonella larvae and caused mortality of Acheta domesticus imagines (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact-intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing the 12 and 40% of mortality at a concentration of 1 mg/mL. TeA was cytotoxic to Sf9 cell line (IC50 25 µg/mL). Thus, model insect G. mellonella and cell line Sf9 could be used for a further toxicological characterization of TeA.

Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism

The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

Mycotoxin Profile and Phylogeny of Pathogenic Alternaria Species Isolated from Symptomatic Tomato Plants in Lebanon

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani, A. alternata, and A. arborescens. A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A.limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg−1, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.