Population Structure And Toxigenicity Profiling Of Aspergillus Section Nigri On The Surface Of Some Of The Main Grape Varieties In Hunan Province

Aspergillus section Nigri is A group of Aspergillus according to morphological classification standards,It was originally widely studied because many species belong to Aspergillus section Nigri has produced a variety of enzymes and is widely applied to the industrial production of citric acid,Later,it was found that many strains belong to Aspergillus section Nigri could produce such mycotoxins as Ochratoxin A(OTA)and Fumonisin B₂(FB₂).Grape and its products are one of the main pollution objects of Aspergillus section Nigri,and it has been reported as the main source of OTA contamination in wine,a few studies have shown that some species belong to Aspergillus section Nigri are related to grape sour rot and Aspergillus black rot.In this study,samples were collected from grapes in the ripening in Hunan province in 2018 and 2019,Aspergillus section Nigri were separate from surface of the samples and investigated the isolation frequency.The study of number of species and population structure of Aspergillus section Nigri in Hunan province were statistics and analyzed by based on the Calmodulin gene sequence or metagenomes with high-throughput sequencing.The toxigenicity of some strains were checked by Ultra performance liquid chromatography-tandem mass spectrometry(UPLC/MS/MS).This study enriched the related research of Aspergillus section Nigri in China and provide a reference for evaluating the occurrence of Aspergillus section Nigri and its toxigenicity on the surface of grape in China.The main findings are as follows:1.In this study,the isolation frequency of Aspergillus section Nigri on 2820 grape fruits of 5 cultivars collected from four regions of Hunan province was investigated.The results showed similar isolation frequency in 2018 and 2019,65.16%and 63.13%respectively.The isolation frequency of Aspergillus section Nigri was slightly higher than other countries.In2018,there were significant differences in the isolation frequency among different regions.In 2019,except for Changde and Changsha,there were no significant differences among other regions.There were significant differences among different varieties in the same area.The temperature one month before sampling had a significant correlation(p<0.05)with the isolation frequency of Aspergillus section Nigri of grape surface,and the precipitation had a extremely significant effect(p<0.001).2.A total of 9 species belong to Aspergillus section Nigri were isolated and identified by based on the Calmodulin gene sequence form the isolate strains in Hunan,which were A.tubingensis?A.nige?A.welwitschiae?A.brunneoviolaceus?A.aculeatinus?A.aculeatus?A.japonicus?A.indologenus?A.uvarum,there are 10 haplotype.The dominant species was A.tubingensis and there were two haplotypes in A.brunneoviolaceus.A total of 2921 strains belong to Aspergillus section Nigri were isolated and identified,which belonged to 3Aspergillus clades.It is A.tubingensis clade,A.aculeatus clade and A.welwitschiae clade.A.tubingensis clade had the largest number of strains.Among the four regions of Changde,Changsha,Yueyang and Huaihua,there are 8 species in total in Huaihua,which the number of species is the most.The number of strains which have possible to produce mycotoxin from Changsha area was the highest,and the structure of the species and clade were significantly different.3.A total of 19 species of fungi and 5 species of Aspergillus section Nigri were identified by metagenomes with high-throughput sequencing.The species belong to Aspergillus section Nigri was A.tubingensis?A.aculeatus?A.welwitchiae?A.niger and A.luchuensis.Comparing the result by metagenomic with the separation method,it showed significant difference in population structure,but the types and proportions of clade are partly consistent,and should be further explored based on a more complete fungal gene pool.5.The 426 strains of A.tubingensis,A.welwitschiae and A.niger were detected for toxigenicity in two years.There were not one strains to be found OTA-producing in two years,but a total of 210 strains produced FB₂.Two mycotoxins were not detected in strains of A.tubingensis.All strains of A.niger could produce FB₂,and the average toxic production was 4586.62 ng/g and 6939.19 ng/g in two years,respectively.The toxigenic proportion of strain A.welwitschiae was as high as 76.47%and 85.48%,and the average toxigenic production was 5622.38 ng/g and 9961.05 ng/g,respectively.The average toxigenic capacity of A.welwitschiae was higher than that of A.niger.The strains of the two species were more widely distributed at both ends of the distribution range.