Microbial Diversity Of Yellow Sun-cured Tobacco Leaves And Application Of Functional Strains To Tobacco Leaves During Curing

Yellow sun-cured tobacco is a specific tobacco plant in China,and has been cultivated for several hundreds of years.Yellow sun-cured tobacco is a raw material of high quality for the development of unique flavor and characteristics of Chinese style cigarette.The mature fresh tobacco leaves have a high content of moisture,and the chemical composition of the leaves is not harmonious.After harvested,it is necessary to cure the fresh tobacco leaves to remove the moisture and unpleasant smell,thus ensuring the tobacco’s preservability.In the curing process of tobacco leaves,a large number of microorganisms still exist in the surface or tissue of tobacco leaves.The metabolic activities of these microorganisms have important effects on the transformation of various chemical components and the quality of tobacco leaves.Compared with the tobacco leaf aging process,the time of curing is relatively short.The research of microbial community structure and diversity of curing tobacco leaves,especially the yellow sun-cured tobacco,is relatively less.On the other hand,the nicotine content of yellow sun-cured tobacco leaves is relatively high,which reduces the availability and economic value of the tobacco leaves.The nicotine degrading microorganism has showed a good application prospect in tobacco waste treatment and aging processing of tobacco leaves.However,there are few reports on the application of tobacco in the curing process.In this thesis,yellow sun-cured tobacco named Yunshai No.1 was studied.High-throughput sequencing(454-pyrosequencing)and pure cultivation methods were used to systematically analyze the composition and structure of microbial community in the tobacco leaves during curing.At the same time,microorganisms capable of degrading nicotine were screened and applied to the curing process of yellow sun-cured tobacco leaves.(1)The bacterial diversity of yellow sun-cured tobacco leaves during curing process was estimated using 454 pyrosequencing technology.The bacterial diversity was low at the initial curing stage,and increased gradually along with the curing process.It indicated that new bacterial groups had been continuously introduced into the tobacco leaves during curing.Except for unclassified bacteria,all OTUs were classified into 14 phyla,among which Proteobacteria were the dorminant group.During curing,the relative abundance of Proteobacteria decreased,while the relative abundance of other groups increased.At the genus level,all OTUs were assigned into 147 genera excluding unclassified bacteria.Pseudomonas and Acinetobacter were the dominant genera throughout the curing process.Using pure cultivation method,358 strains of bacteria were isolated from yellow sun-cured tobacco leaves during curing.The number of bacteria isolated in the initial stage of curing was less,but in the middle and the end of curing,the number of bacteria increased.Based on the morphological characteristics and phylogenetic analysis of 16S rRNA sequences,358 strains were classified into 4 phyla,13 families,14 genera and 39 species.Firmicutes was predominant,and accounted for 62.57%.At the genus level,48.60%of the bacterial strains belonged to the genus Bacillus,13.13%belonged to the genus Pseudomonas.(2)The fungal diversity of yellow sun-cured tobacco leaves during curing process was evaluated using 454 pyrosequencing technology.The difference of diversity index between the initial and end of the curing of the lower and middle tobacco leaves was not significant.The fungal diversity of upper leaves at the end of curing was slightly higher than that at the beginning stage.The composition of fungal community changed during curing.About 42%of the fungal groups disappeared during curing,but at the same time,there were about 36%new fungal groups introduced into tobacco leaves.Except for unclassified bacteria,all OTUs were classified into 3 phyla,6 classes,19 orders,28 families,59 genera.Ascomycota and Epicoccum were the dominant groups at the levels of phylum and genus,respectively.126 strains of fungi were isolated from the tobacco leaves.These strains were classified into 2 phyla,5 classes,10 orders,14 families,17 genera.Diversity analysis showed that culturable fungi group was abundant.Ascomycota was the dominant phylum.Epicoccum,Arthrinium and Fusarium were the dominant genera,accounting for 23.02%,13.49%and 11.90%,respectively.(3)Based on the selective medium in which nicotine was the sole carbon and nitrogen source,39 strains of nicotine degrading bacteria were isolated from rhizosphere soil,roots,stems and leaves.According to morphological identification and phylogenetic analysis of 16S rRNA sequences,the 39 strains of nicotine degrading bacteria were classified into 9 genera,namely Pseudomonas,Arthrobacter,Achromobacter,Agrobacterium,Ochrobactrum,Stenotrophomonas,Klebsiella,Bacillus and Cellulosimicrobium.The number of Pseudomonas and Arthrobacter strains was predominant among all the isolates.Among the four kinds of samples,the number of isolates from rhizosphere soil was the highest.(4)The strain L16 which was isolated from tobacco leaves was selected to evaluate the effect on nicotine and other chemical components during curing of yellow sun-cured tobacco leaves.Through morphological,physiological and biochemical tests,and phylogenetic analysis of 16S rRNA sequences,strain L16 was identified as Pseudomonas sp.L16.The optimum incubation temperature of Pseudomonas sp.L16 was 28℃,nicotine concentration of 1.0 g/L～1.5 g/L,pH6.0～7.0.Psudomonas sp.L16 was applied on the fresh leaves of yellow sun-cured tobacco.After curing,the nicotine contents of the three leaf positions were all significantly decreased.The total sugar,reducing sugar,sugar nicotine ratio and nitrogen nicotine ratio were increased in different degrees.The internal chemical composition of tobacco leaves was more coordinated,the main aroma quality was improved,the cigarette vigour was more moderate,and the overall balance and coordination was obviously improved.