Metabolic Pathway Analysis Of FBMN On The Virulence Of Different Alternaria Spp. And The Infection Of Laiyang Pear

Alternaria spp.is one of the most widely distributed fungi in the world.It exists on different substrates in nature,including saprophytic fungi,endophytes and pathogenic bacteria.More than 95 % species parasitized on plants,causing a variety of plant diseases.Although Alternaria spp.is an asexual fungus,it can not only exist asexually,but also reproduce sexually to produce new Streptomyces and new metabolites.More than 70 metabolites have been proved to have obvious toxicity,such as teratogenicity,lethality,carcinogenicity,mutagenicity,cytotoxicity,acute toxicity,and embryonic toxicity.Due to environmental reasons,these metabolites may accumulate in fruits,vegetables,products and agricultural products,and then flow into the market,which seriously threatens human health.At present,the studies on Alternaria spp.in China and abroad have mainly focused on analysis of free Alternaria toxins,while there are relatively few studies on the masked toxins and metabolic pathways of Alternaria.Therefore,this paper uses a variety of visual data analysis software and methods to analyze the metabolic pathways of free and masked toxins in Alternaria spp.In the first part,five strains of pathogens of pear black spot and two strains of endophytic fungi in cherry were selected as experimental materials,and were cultured in Potato carrot agar,Potato dextrose agar and Potato sucrose agar,respectively.Metabolite analysis was performed using UPLC-MS/MS.Nine free mycotoxins were confirmed by comparison and identification with commercial toxin standards as well as the results of previous laboratory studies,which were alternariol,alternariol monomethyl ether,altenuene,altenusin,tentoxin,tenuazonic acid,and altertoxin-I by comparison with the standard toxin.Dehydroaltenusin and desmethyldehydroaltenusin;four sulfate-binding toxins were AOHSulfated,AME-Sulfated,DHA-sulfated and DMDA-Sulfated,respectively.Using the response value of high-resolution mass spectrometry as the quantitative standard,the heat map was made.Toxin content analysis of different strains at different culture times in different media showed that the amount of streptavidin produced was the most on PSA medium and the toxin production was the least in PCA medium.MZmine 2 and Global Natural Products Social Molecular Networking analysis tools were used to analyze the metabolic pathways of streptomycin in three media.Metadata files were used to analyze the analysis network,and a total of six Streptomyces mycotoxin metabolic clusters were found.The mold cultured in PSA medium had relatively complete metabolic pathways.In the second part,the Laiyang pear was used as the experimental material in this experiment.Seven strains of Alternaria were resuscitated to PSA medium and the mycelia were prepared and inoculated on the Laiyang pear fruit.The inoculated samples were stored at 4 °C and 25 °C,respectively.The infection process of strains on Laiyang pear under different temperature environments was simulated to explore the metabolic pathway and pollution degree of Alternaria toxin in Laiyang pear fruit.UPLC-MS/MS was used to analyze the samples extracted from the lesion.The experimental results showed that Alternaria toxins were detected in the infected Laiyang pear fruits at 4 °C and 25 °C,which were identified as seven free toxins(AOH,AME,ALT,ALU,TEN,Te A and ATX-I)and three glucosed covert toxins(AME-Glucosed,ALU-Glucosed and Te A-Glucosed).Among them,Te A had the highest content of 358.099 mg/kg.FBMN was used to establish molecular networks for confirmed free and covert toxins,and 268 compounds were matched in the database by comparing with the spectral database of GNPS platform.Molecular networks show that AOH,AME,ALT,ALU,TEN,Te A and Te A-G all have metabolic pathways,while glucose-concealed toxins of ATX-I,AME and ALU are isolated in the metabolic pathway diagram due to the small number of spectral nodes,resulting in non-corecognition with the spectral database.It was found that strain 122 did not produce ALU in the mediums,but it was detected in the four extraction samples of Laiyang pear tissues infected at 25 °C.This was because the signal molecules or secondary metabolites produced by the host could promote or inhibit the toxin production of fungal strains in the process of toxin production by the strain infecting plants,resulting in a certain difference in the toxin production types of the same strain in artificial medium and plants.