Study On Effect Of Exogenous Substances On The Formation Of Green Spots And Physiology Quality Of Cigar Leaves At Air-curing Stage

In China,cigar is not only cultivated in a short period of time,but also it has a small cultivation area,which makes most of the high-quality raw materials for cigars depending on imports.With the rapid growth of the demand for cigar tobacco in Chinese market,it is of great theoretical and practical significances to carry out the research on the air-curing technology of high-quality raw cigar tobacco and its quality and physiological change mechanism.Cigar wrapper varieties have high requirements for appearance,and the"green spots"in the cigar wrapper varieties appearing during the air-curing period will seriously affect the quality.In this study,the cigar coating variety CX-010（Chu Xue 10）was used as the experimental material at the base of Hubei Cigar Tobacco Research Institute.Based on the study of the physiological mechanism of"green spots"formation,the effects of spraying sucrose and other chemical substances on the formation of"green spots",water and pigment content,and quality of air-curing tobacco leaves were investigated.At the same time,the effects of spraying chemical substances on the air-curing quality of tobacco leaves and their physiological mechanisms were studied by means of non-targeted metabolomics and microstructure observation.The aim is to provide a basis for the large-scale production of high-quality cigar tobacco leaves in Hubei tobacco areas.The main results obtained are as follows.1.Incomplete decomposition of chlorophyll in cigar wrapper variety CX-010 leaves during air-curing was the main reason for the formation of"green spots".The chlorophyll content of the green spots tissues was about twice as much as that of the brown tobacco tissues,and there was no significant difference in carotenoid content.The ultrastructure of air-curing tobacco tissues showed that the lipid morphology of the green spots tissues was different from that of the brown tobacco tissues,and the organelle structure still existed in some cells.A total of561 differentially abundant metabolites（DAMs）（VIP>1,P<0.05）was detected in green spots and normal brown air-curing tissues,and the most significant differences were found between the two tissues in the KEGG（Kyoto Encyclopedia of Genes and Genomes）metabolic pathway related to phenylpropanoid biosynthesis（P<0.001）and autophagy（highest metabolite enrichment）differences.The DAMs such as Delphinidin,4-Coumaric acid,Acetyl-L-tyrosine,4-Guanidinobutanoic acid,and N2-Acetylornithine was significantly lower in green spots air-curing tobacco tissue than in normal-air-curing tobacco tissue sites,and unsaturated fatty acids were significantly higher than in normal-curing tobacco tissue sites.The physiological process of air-curing tobacco leaves with disturbed lipid metabolism and amino acid metabolism led to incomplete degradation of proteins and pigments,which eventually formed"green spots".The high humidity in the air-curing room during the pre-air-curing period of tobacco leaves may be an external environmental factor for the formation of"green spots".2.Spraying exogenous substances affected the number of"green spots"and chemical quality of air-curing tobacco leaves in different degrees.The chlorophyll and carotenoid degradation rates of sucrose and chitosan treatments were not significantly different in the first 10days of air-curing,being about 80%and 60%,respectively,but the chlorophyll content of sucrose treatment was almost all slightly lower than that of chitosan oligosaccharide and control treatment in the same period.The quality of tobacco leaves sprayed with sucrose treatment was better,which showed a reduction in"green spots"in appearance and a significantly higher content of total sugars and reducing sugars than those sprayed with chitosan oligosaccharide and distilled water,which may contribute better to the aroma of the smoke;the nicotine content was also significantly lower compared with the distilled water treatment,and the nicotine content decreased significantly with increasing sugar concentration（P<0.05）,with the highest in the S5（0.5 M sucrose）treatment,reaching 32.2%.Compared to the control treatment,there was also no significant effect on changes in potassium,chloride,and polyphenol content in the sucrose-treated tobacco leaves.The tobacco leaves treated with chitosan oligosaccharide were not significantly reduced in appearance for“green spots”,and the intrinsic quality was significantly higher in total sugar and reducing sugar content than the distilled water treatment,but lower than the sucrose treatment;nicotine content was also significantly lower compared to the control,but higher than the sucrose treatment.Compared to the control,the chitosan oligosaccharide-treated tobacco leaves were not significantly altered for potassium and chloride content,but both were within the appropriate range.The chemical quality of tobacco leaves sprayed with sucrose after 2 d of wilting did not differ significantly from that of the direct sprayed sucrose treatment,but the period of the fastest degradation rate of substances was 1-2 d earlier,and the effect on the management of“green spots”was stronger.However,cigar tobacco leaves sprayed with plant growth regulators showed a slower rate of yellowing and browning overall,with the control treatment leaves having completely browned by the 15^th day of air-curing,while a larger area of yellow-green tobacco leaves still existed in the trans-zeatin concentration gradient treatment.In summary,among the exogenous spray sucrose,chitosan oligosaccharide,naphthalene acetic acid,indole butyric acid,and trans-zeatin,the spraying of sucrose had the best effect on alleviating the formation of"green spot"in air-curing tobacco leaves.Considering the formation of“green spot”and chemical quality of air-curing tobacco,the treatment of 0.4 M sucrose spraying concentration was the best,and the effect of spraying after wilting was better than that of direct spraying.3.Exogenous sucrose significantly affected the abundance of carbon and nitrogen metabolites and metabolic pathways in air-curing tobacco leaves,thus resuting in the formation of"green spots".All sucrose treatments alleviated the green spot phenomenon,and S3（0.4 M sucrose）had the fewest“green spots”.Thus,the tobacco leaves of the S3 and CK（water）treatments at two air-curing stages were used to perform metabolomics analysis with non-targeted liquid chromatography-mass spectrometry to determine the physiological mechanism.A total of 259 and 178 differentially abundant metabolites（DAMs）between S3 and CK-treated tobacco leaves were identified in the early air-curing and the end of air-curing stages,respectively.These DAMs mainly included lipid and lipid-like molecules,carbohydrates,and organic acids and their derivatives.Based on the KEGG pathways analysis,the S3 treatment significantly altered carbohydrate metabolism（pentose phosphate pathway,sucrose and starch metabolism and galactose metabolism）and amino acid metabolism（tyrosine metabolism and tryptophan metabolism）in air-curing tobacco leaves.Sucrose treatments alleviated the formation of"green spots"by changing the abundance of metabolites related to chlorophyll decomposition on the one hand,and provided the energy required for chloroplast degradation to alleviate the appearance of"green spots"in air-curing tobacco leaves on the other hand,which may be the physiological mechanism of sucrose spraying to alleviate the formation of"green spots".In conclusion,the green spots were caused by incomplete chlorophyll degradation during air-curing and ineffective control of air-curing room temperature and humidity.In this study,the spraying of sucrose was effective in controlling green spots,and the best treatment was 0.4 M sucrose after 2 d of wilting,which could improve not only the appearance but also the chemical quality of CX-010 cigar leaves.