Research On The Biological Diversity Of Aspergillus Flavus Isolated From Stored Paddy In Southern China And Aflatoxin Biocontrol Technology

Paddy rice is one of the staple foods in southern China,where regions are characterized by high temperatures and humid conditions,the favorable environment can significantly enhance the infection of Aspergillus flavus and aflatoxins accumulation,thus posing a threat to people’s health.Therefore,the study was researched on the distribution and aflatoxin production of A.flavus isolated from stored paddy in southern China and revealed the relationship with ecological environment.The biodiversity of A.flavus isolates was studied in order to reveal the effect of diversity on A.flavus population.Finally,the inhibitory effect of atoxigenic A.flavus on toxigenic A.flavus was studied.The main results of this paper are as follows:1.A total of 315 rice grain samples were collected from major rice-producing provinces（Jiangsu,Zhejing,Hunan,Hubei,Anhui,Jiangxi,and Fujian）in southern China.Then strains were isolated and purified,based on morphological characteristics and Ca M sequencing,81 A.flavus strains were isolated and identified,most of A.flavus isolates originated from Hunan and Jiangxi,and fewer A.flavus isolates were collected in Jiangsu and Zhejiang.HPLC-FLD results showed that 56 out of the 81 A.flavus isolates produced detectable levels of AFB₁.The distribution of toxigenic and atoxigenic A.flavus isolates exhibited considerable variation across seven provinces,as the temperature decreased in the seven provinces included in the study,the percentage of toxigenic A.flavus isolates gradually increased.The distribution and AFB₁production of A.flavus isolates were affected by geographical region and temperature and humidity.2.By measuring the sclerotium size of A.flavus isolates found that the S-type（sclerotia<400μm in diameter）A.flavus isolates produced high level of AFB₁,while L-type（sclerotia>400μm in diameter）A.flavus isolates produced no detectable AFB₁or low levels of AFB₁.PCR-based molecular approaches were used to identify the presence of AFB₁ biosynthetic gene in A.flavus isolates,amplified afl R PCR products were obtained from all toxigenic A.flavus isolates,while fewer or no afl R PCR products were obtained from atoxigenic A.flavus isolates.Genetic diversity analysis using inter-simple sequence repeats（ISSR）markers revealed significant genetic differences among the isolates,the genetic similarity coefficients ranging from 0.53 to 1.00.The dendrogram produced 7 main clusters using UPGMA,delineated at a genetic similarity coefficient value of 0.61.The co-culture experiments of toxigenic A.flavus isolates and atoxigenic A.flavus isolates from the same grain depot showed that the existence of atoxigenic A.flavus isolates could reduce the risk of AFB₁ contamination during storage in the grain depot.3.The inhibition rule of atoxigenic A.flavus against toxigenic A.flavus was studied and results showed that atoxigenic A.flavus could reduce the production of AFB₁ by inhibiting the sporulation and the radial growth of toxigenic A.flavus.In the co-culture experiment of single atoxigenic A.flavus and toxigenic A.flavus,the inhibition effect of atoxigenic A.flavus was the best when the inoculation of atoxigenic A.flavus was delayed for 1 day at the spore concentration ratio of 1:50.However,in the co-culture experiment of mixed atoxigenic A.flavus and mixed toxigenic A.flavus,the inhibition effect reached the best when the mixed atoxigenic A.flavus and toxigenic A.flavus were inoculated at the same time with the spore concentration ratio of 1:2.In situ inoculation,rice and peanut were used as the culture substrate,atoxigenic A.flavus showed inhibitory effect on the growth and the AFB₁ production of toxigenic A.flavus.Our results indicated that few atoxigenic A.flavus could have a better biological control effect on toxigenic A.flavus,which provided a theoretical basis for the prevention and control of aflatoxin pollution.