To investigate the effects of optimizing basal-to-topdressing ratios combined with nitrogen application rates on nitrogen metabolism and nitrogen uptake/utilization in flue-cured tobacco,a field experiment was conducted using the cultivar Yueyan 97.Five treatments were designed:conventional basal-to-topdressing ratio (6∶4)+conventional nitrogen rate (CK),basal-to-topdressing ratio (4∶6)+conventional nitrogen rate (T1),basal-to-topdressing ratio (4∶6)+10% nitrogen reduction (T2),basal-to-topdressing ratio (4∶6)+20% nitrogen reduction (T3),and basal-to-topdressing ratio (4∶6)+30% nitrogen reduction (T4).It analyzed the impacts of these treatments on nitrogen metabolism,photosynthetic characteristics,dry matter accumulation,nitrogen uptake,nitrogen use efficiency,and nitrogen balance.The results showed that after optimizing the basal-to-topdressing ratio to 4∶6,the activities of key nitrogen metabolism enzymes,including glutamate dehydrogenase (GDH),aspartate transaminase (AST),and ferredoxin-glutamate synthase (Fd-GOGAT),were all higher than those under the traditional basal-to-topdressing ratio of 6∶4.When combined with nitrogen reduction treatments,the activities of nitrogen metabolism enzymes decreased.Specifically,T2 (10% nitrogen reduction) showed no significant difference from CK,while T3 and T4 (20%—30% nitrogen reduction) exhibited marked declines in enzyme activity.Optimizing the basal-to-topdressing ratio to 4∶6 also improved photosynthetic rate,SPAD values,dry matter accumulation,and nitrogen accumulation,with significant differences observed during the late growth stages.Furthermore,the optimizing 4∶6 ratio significantly enhanced nitrogen use efficiency.Compared to CK,nitrogen agronomic efficiency,partial factor productivity,recovery efficiency,and economic utilization efficiency increased by 20.68%,6.89%,4.06 percentage points,and 3.78 percentage points,respectively.Nitrogen reduction treatments reduced soil nitrogen surplus by 17.04—33.36 percentage points compared to CK.In conclusion,optimizing the basal-to-topdressing ratio from 6∶4 to 4∶6 in southern tobacco-growing regions enhances nitrogen metabolism during mid-to-late growth stages,improved photosynthetic performance,promotes dry matter and nitrogen accumulation,and increased nitrogen use efficiency.Coupled with 10% reduction in fertilizer can also reduce fertilizer input,mitigetes soil nitrogen surplus,and prevent environmental risks.

In order to clarify the synergistic effect of trehalose alcohol,phosphate-solubilizing microorganisms and trace elements on the yield and quality of flue-cured tobacco,a field experiment was carried out with Yunyan 87 as the test material.The control(S1),trehalose alcohol(S2),trehalose alcohol+compound phosphate-solubilizing bacteria(S3),trehalose alcohol+compound phosphate-solubilizing bacteria+compound trace elements(S4)were set up to study the effects of different fertilization treatments on agronomic traits,conventional chemical components and aroma substances of flue-cured tobacco.The results showed that compared with S1,S3 and S4 could improve the effective leaf number and leaf width of flue-cured tobacco.S4 treatment was more helpful to coordinate the chemical composition of cured tobacco leaves.The contents of total sugar,total nitrogen and nicotine were increased by 12.18%,70.00% and 20.30%,respectively,and the contents of aroma substances such as carotenoids,phenylalanines and brown-reacting species.The results of comprehensive index analysis showed that the comprehensive indexes of S1-S4 treatments were 21.06,23.22,24.66 and 27.08,respectively,and the comprehensive indexes were ranked as S4>S3>S2>S1,indicating that the comprehensive performance of flue-cured tobacco under the treatment of trehalose alcohol combined with trace elements and microbial agents was the best,indicating that the synergistic effect between trehalose alcohol,phosphate-solubilizing bacteria and compound trace elements was obvious,which could be used as the optimal formula of trace elements and microbial special fertilizer for tobacco leaves.

The biological mechanism of Streptomyces TOR3209 volatile organic compounds(VOCs)promoting tobacco growth and development was studied.The growth-promoting properties of VOCs in the fermentation broth were identified by two separate plates,and the VOCs components were analyzed by solid phase microextraction and gas chromatography-mass spectrometry.VOCs of Streptomyces TOR3209 at different concentrations could promote the growth of tobacco,and the effect of adding 5 mL(6×10⁷ CFU/mL)strain fermentation liquid was the most significant in the treatment group,which increased 238% compared with the control group.A total of 32 substances were extracted from the VOCs of Streptomyces TOR3209 fermentation broth,and the growth-promoting function of dinbutyl adipate was verified.The results showed that dinbutyl adipate of different mass concentrations could promote the growth of tobacco,and the growth promoting effect of 1 mol/mL was the most significant.Through transcriptome analysis,it was found that many genes such as laccase were up-regulated in the tobacco treated group compared with the control group.VOCs of Streptomyces TOR3209 had a significant growth promoting effect on tobacco,and dinbutyl adipate regulated several growth promoting genes in tobacco.This study clarified the mechanism of the VOCs of Streptomyces TOR3209 regulating tobacco growth and development,and provided a theoretical basis for the development of efficient and environmentally friendly microbial production promotion products and the application evaluation.

In order to explore the diurnal variation characteristics and influencing factors of surface CO₂ and N₂O emission fluxes in tobacco fields in central Henan under the management mode of continuous green manure incorporation,and to determine the optimal collection time of the two gases,in 2021,based on a long-term experiment in Maozhuang Science and Education Park of Henan Agricultural University,a 24-hour continuous dynamic observation was carried out by static dark box-gas chromatography every 30 days after tobacco seedlings were transplanted.The daily emission fluxes of CO₂ and N₂O in tobacco fields under nitrogen phosphorus potassium treatment(NPK)and NPK+planting and turning over ryegrass treatment(NPKG)were measured.The results showed that the tobacco field soil in central Henan was the source of CO₂ and N₂O emissions,and the emission trend was similar to the trajectory of atmospheric temperature change,which was high in day and low in night,and the average emission flux of day and night reached a significant difference level.The CO₂ emission flux showed an inconspicuous bimodal pattern at 30 and 60 days after transplanting,and a single peak state of high day and low night at 90 days after transplanting.The N₂O flux showed a single peak state of high day and low night.The CO₂ and N₂O emission fluxes of NPKG treatment were significantly higher than those of NPK treatment.The CO₂ emission fluxes of three typical days were 90 d>60 d>30 d,and the N₂O emission fluxes were 60 d>90 d>30 d.During the observation days,water-filled porosity of soil(WFPS) and 10 cm soil temperature jointly affected the emission rates of CO₂ and N₂O,and the 10 cm soil temperature was significantly or extremely significantly positively correlated with the daily emission fluxes of CO₂ and N₂O.The correction coefficients of daily CO₂ and N₂O emission fluxes at 9:00 and 21:00 were the closest to 1,and there was no significant difference compared with the daily average emission flux.There was a significant positive correlation between CO₂ flux and N₂O flux.Within a certain range,the N₂O flux increased linearly with the increase of CO₂ flux.In summary,continuous green manure returning increased the surface CO₂ and N₂O emission flux of tobacco field,and the best collection time of CO₂ and N₂O was around 9:00 and 21:00.During the three sampling days of tobacco growth period,the surface CO₂ emission flux of NPK treatment and NPKG treatment was the highest at 90 days after transplanting,followed by 60 days.The N₂O emission flux was the highest at 60 days after transplanting,followed by 90 days.

In order to investigate the effects of endophytic fungus Piriformospora indica on the rhizosphere microenvironment of tobacco,a pot experiment was conducted under greenhouse conditions,using Yunyan 87 as the experimental material to analyze its effects on tobacco root exudates,rhizosphere soil enzyme activities and bacterial diversity through non-targeted metabolomics,soil enzyme activity measurement,high-throughput sequencing,and microbial community functional prediction.The results showed that the P.indica successfully colonized in the roots of tobacco,increasing the content of 18 different root exudates,mainly including acids,esters,alcohols,terpenoids,and phenolic compounds,significantly enriching the metabolism pathways of L-phenylalanine,taurine and hypotaurine,and L-tryptophan.Compared to using sterile water as a control,P.indica increased the activities of urease,sucrase,and alkaline phosphatase in tobacco rhizosphere soil,with urease and sucrase activities reaching significant levels.P.indica increased the richness and diversity of bacterial communities in tobacco rhizosphere soil,with a significant increase in Chao1 index and Observed_otus index comparing with the control.This altered the bacterial community structure at the phylum and genus levels,and increased the relative abundance of dominant phyla of Acidobacteriota and Actinobacteriaota,as well as Pseudomonas and Gemmatimonas genera.It also increased the gene abundance involved in carbohydrate metabolism and translation pathways in rhizosphere soil bacteria,which had a certain impact on bacterial community function.It indicates that P.indica could improve the rhizosphere microecological environment of tobacco,providing a theoretical basis for understanding the mechanism of their promotion of tobacco growth and tobacco planting production.

In order to reveal the expression patterns of PsbS protein in Nicotiana tabacum,the full-length NtPsbS gene sequence was cloned from cDNA of the tobacco cultivar K326,and the multiple protein sequences of NtPsbS gene and PsbS genes of seven crops including rice,tomato and soybean were compared by DNAMAN software.The phylogenetic analysis was carried out by MEGA 11 software,and the phylogenetic tree was established by neighbor-joining method.Tissue expression of NtPsbS gene in tobacco at different growth stages was detected by qRT-PCR.The plant expression vector pS1300-PsbS-GFP was constructed to find subcellular localization of the NtPsbS mature protein.Finally,the mRNA levels of NtPsbS under abiotic stresses were analyzed in K326.The results showed that the total length of tobacco NtPsbS gene was 825 bp,encoding 274 amino acids.The homology of tobacco NtPsbS protein and tomato SlPsbS protein was the highest up to 91%.The NtPsbS gene exhibited the highest mRNA levels in the leaves among the tissues,including leaves,roots,stems,seeds and other parts of K326.The mature NtPsbS protein was located in chloroplasts.For the abiotic stress,it was found that the expression level of NtPsbS was significantly increased under salt stress,cold stress and Abscisic acid(ABA)treatments.In summary,the expression level of NtPsbS was the highest in tobacco leaves at different growth stages,and it was higher when under the treatments of salt stress,cold stress and ABA,indicating that this gene may be involved in the salt and cold stress resistance and ABA metabolic pathway in tobacco,which could provide basis and reference for the functional analysis of the NtPsbS gene in future.

In order to provide production advice of high-quality upper tobacco leaves,field experiment was carried out to elucidate the effects of nitrogen application on the growth,development,carbon and nitrogen metabolism of upper leaves of different tobacco varieties.Three treatments,37.5(LN),75.0(MN)and 112.5 kg/ha nitrogen application rate(HN)were conducted with NC89 and Yunyan 87 as the test varieties.The effects of different treatments on the agronomic traits,photosynthetic characteristics,leaf tissue structure,key enzymes activity of carbon and nitrogen metabolism,and chemical composition of the upper leaves of were studied.The results showed that the leaf length,leaf width,leaf area,and leaf dry mass of the upper leaves of both varieties were significantly improved with the increased of nitrogen application rates.At 115 days after transplanting(DAT),the leaf area of NC89 and Yunyan 87 of HN were significantly increased by 63.10% and 68.43% compared to LN,respectively.Increasing nitrogen rate improved the leaf chlorophyll content of NC89,the leaf chlorophyll content of HN was 6.67%—37.50% higher than that of LN,respectively.The leaf net photosynthetic rate was notably improved by increasing nitrogen rate of Yunyan 87,especially at 70 DAT and 80 DAT.The leaf palisade tissue,leaf spongy tissue,and leaf thickness were markedly promoted with the increased nitrogen application rate on 85—115 DAT.The palisade tissue and spongy tissue thickness of LN and MN were almost stable from 95 to 115 DAT,but HN improved by 9.82%—14.08% and 10.72%—13.72% respectively.The leaf carbon content and leaf carbon and nitrogen ratio of both varieties were significantly decreased by reduced nitrogen rate,while leaf nitrogen content significantly increased.The activities of invertase,sucrose phosphate synthase,sucrose synthase,and glutamate synthase of both varieties were increased with the increased nitrogen application rate.HN decreased the amylase activity of Yunyan 87,but increased that of NC89.At 115 DAT,the amylase activity of HN of Yunyan 87 was 27.53% lower than MN,while HN increased amylase activity by 33.86% and 21.74% compared to LN and MN of NC89,respectively.The activity of glutamine synthetase of Yunyan 87 significantly increased with the increased nitrogen application rate,while there was no significant difference among different treatments of NC89.The content of flue-cured tobacco leaf reducing sugar and total sugar were reduced,but the content of nicotine and total nitrogen were improved with increased nitrogen application rate.Under the same nitrogen application rate,the content of flue-cured tobacco leaf nicotine,total nitrogen,and potassium of Yunyan 87 were higher than NC89,while the content of reducing sugar,content of total sugar(except LN),sugar and nicotine ratio,and nitrogen and nicotine ratio were lower than NC89.The effect of nitrogen application rate on upper leaves of tobacco varieties is difference.Increasing nitrogen application rate could promote the growth,development and carbon metabolism,reduce sugar and nicotine ratio and nitrogen and nicotine ratio,and improve the coordination of chemical components of NC89.However,increasing nitrogen application rate could lead to excessive nitrogen metabolism,delay the transformation from nitrogen metabolism to carbon accumulation metabolism,and cause late maturity of Yunyan 87.

To study the effects of ridge height on soil heat status,root growth,and tobacco maturity tolerance in tobacco planting,a 2-year field experiment was conducted using the flue-cured tobacco variety Yueyan 97 as the test material from 2022 to 2023.Three treatments were set up,including a ridge height of 30 cm(CK),a ridge height of 38 cm(T1),and a ridge height of 46 cm(T2),to analyze the changes in soil temperature and heat flux,root appearance and growth indicators,root vitality,and tobacco maturity related indicators under different ridge heights.The results indicated that the daily temperature difference in soil varies with the depth of the soil layer,and the daily temperature difference in the surface layer of the soil was the largest.Increasing ridge height can increase the average temperature of tobacco planting soil by 0.4-1.8 ℃ and increase soil heat flux by 4-56 W/m².In the 2 a experiment,the maximum increase in root length for a ridge height of 38 cm compared to a ridge height of 30 cm was 27.26%,the maximum increase in root dry mass was 26.21%,and the maximum increase in root vitality was 14.97%,with significant differences.Compared with a ridge height of 30 cm,the soluble protein content,peroxidase activity,and cell membrane stability index of a ridge height of 38 cm increased by 17.99%,27.82%,and 9.05 percentage points(2022),respectively and by 10.23%,12.44%,and 8.16 percentage points(2023),respectively.The maximum decrease in malondialdehyde content was 24.84% and 44.43%,respectively.Correlation analysis showed that root activity was significantly negatively correlated with malondialdehyde content,peroxidase activity was extremely significantly positively correlated with root growth indexes, and soluble protein content was significantly positively correlated with root length.In summary,increasing ridge height is beneficial for improving the thermal status of tobacco planting soil,promoting root growth,enhancing root vitality,enhancing leaf antioxidant capacity,and enhancing tobacco maturity tolerance.A ridge height of 38 cm is an appropriate ridge height for promoting root growth and improving tobacco maturity tolerance in southern tobacco regions.

To explore the effects of fertilizer reduction and organic fertilizer application on physicochemical properties and microbial community structure of tobacco-planting soil,and provide theoretical reference for fertilizer reduction and rational application of organic fertilizer in tobacco production.Using conventional fertilization without organic fertilizer as control(CK),Illumina high-throughput sequencing technology combined with bioinformatics,the changes of soil physical and chemical properties and microbial community structure under different treatments such as 10% reduction of fertilizer(T1),10% reduction of fertilizer+sesame cake fertilizer(T2),10% reduction of fertilizer + humic acid fertilizer(T3),and 10% reduction of fertilizer + sesame cake fertilizer + humic acid fertilizer(T4)were analyzed.The results showed that compared with CK,soil nutrient content and soil enzyme activity decreased under T1 treatment,and soil physical properties were slightly improved.Combined with organic fertilizer,soil nutrient and physical properties were further improved,bulk density decreased,moisture content and porosity increased,and the contents of available phosphorus and available potassium under T2 and T4 treatment were significantly higher than those under CK and T1 treatment.The enzyme activity of soil treated with organic fertilizer increased significantly.Combined application of organic fertilizer increased the bacteria and fungi in tobacco-growing soil,among which the dominant bacteria were Proteobacteria and Actinobacteriota.Followed by Firmicutes,Bacteroidota,Chloroflexi and Acidobacteriota.The dominant fungal groups are Ascomycota,Anthophyta,Mortierellomycota,Chlorophyta,Ciliophora and Basidiomycota.The Alpha diversity index showed that the reduction of fertilizer decreased the richness of microbial community,but the combined application of organic fertilizer increased the diversity index of bacterial and fungal communities,and the bacterial community richness increased more significantly.RDA analysis showed that the important soil physicochemical factors affecting soil microbial community structure and diversity included organic matter,available potassium,alkali-hydrolyzed nitrogen,available phosphorus and soil physical properties,etc.Soil physicochemical factors had a greater impact on bacterial community structure.In conclusion,under the condition of fertilizer reduction,organic fertilizer combined with organic fertilizer can significantly improve soil nutrients,physical properties,soil enzyme activity and microflora structure,especially when combined with sesame cake fertilizer and humic acid fertilizer treatment.

The WRKY transcription factor family plays an important role in regulating abiotic stress.To systematically analyze the sequence characteristics and expression patterns of NtWRKY11 gene in tobacco,and explore the response mechanism of NtWRKY11 under abiotic stress such as low temperature and drought,the full-length cDNA sequence of NtWRKY11 gene was amplified by PCR using common tobacco cDNA as a template.The basic properties of NtWRKY11 protein were analyzed by bioinformatics software and the subcellular localization of NtWRKY11 protein was studied by constructing a plant expression vector.The expression of NtWRKY11 gene was detected by qRT-PCR technology in different tissues at the full flowering stage and under different abiotic stresses.The results showed that the full-length cDNA of NtWRKY11 gene in tobacco was 999 bp,encoding 332 amino acids,and it shared 54.23% similarity with ramie BnWRKY11.Its promoter region contained three kinds of cis-acting elements(one MBS,one MYB and three ARBE),which probably worked together to enhance the drought resistance of plants.It also contained three salicylic acid-responsive cis-acting elements(TCA-element),which could improve the low temperature tolerance of plants.Subcellular localization results indicated that the NtWRKY11 protein was located in the nucleus.The expression analysis of NtWRKY11 in different tissues at the full flowering stage showed that NtWRKY11 was highly expressed in old leaves,significantly higher than in roots.However,the expression in flowers was significantly lower than that in roots,and there was no significant difference between stems,new leaves and roots. The expression analysis under abiotic stress showed that the relative expression of the gene was significantly higher than that of normal (CK).The relative expression level under high salt stress was not significantly different from CK,while the relative expression level under high temperature stress was significantly lower than CK.All in all,NtWRKY11 is highly expressed in old leaves,and its expression level is enhanced under abiotic stresses such as drought stress and low temperature stress,indicating that this gene acts as a forward transcription factor to regulate drought and low temperature stress.

To investigate the effects of Piriformospora indica combined with Arbuscular mycorrhizal fungi (AMF) on drought resistance of tobacco,Yunyan 87 was inoculated with sterile water (CC),P.indica (CP),AMF (PC),P.indica and AMF (PP),and the contents of proline (Pro),superoxide dismutase (SOD),peroxidase (POD),malondialdehyde (MDA) and the expression of drought-related genes NTNAC1 and NAC4 in tobacco leaves were determined under stress in the form of natural drought.The results showed that P.indica and AMF could promote the growth of above-ground and underground parts of tobacco,and chlorosis of leaves and symptoms of their drought were slight.After 7 days of drought stress,the contents of Pro in tobacco leaves of CP,PC and PP groups were 1.39,1.59 and 1.78 times higher than those of CC in group,respectively.The activities of SOD and POD in tobacco leaves increased first and then decreased.The activities of SOD in CP,PC and PP groups were 1.15,1.22 and 1.33 times higher than those in CC group,POD activity was 1.33,1.46 and 1.85 times higher than those of CC group,respectively.The content of MDA in tobacco leaves was decreased by 21.98%,23.98% and 24.84% in CP,PC and PP groups respectively.The expression levels of NTNAC1 and NAC4 in tobacco leaves were up-regulated.The expression levels of NTNAC1 in CP,PC and PP groups were 3.37,3.88 and 5.07 times higher than those in CC group,the expression levels of NAC4 gene were 3.04,3.59 and 5.56 times higher than those of CC group,respectively.This study indicates that P.indica and AMF showed significant synergistic effects,which could significantly improve the drought resistance of tobacco.

Define the cultivation measures of long-leaf tobacco seedlings in tobacco-rice rotation area to promote the early growth and rapid development of paddy-tobacco.Yunyan 87 was used as the material.The effects of three factors on dry matter and nutrient accumulation and distribution,and fertilizer use efficiency of flue-cured tobacco were studied,namely,the aperture of nursery site(136,200 pores),microbial agent(substrate with Miaoqiangzhuang microbial agent,substrate without microbial agent),and transplanting leaf age(8-leaf transplanting,10-leaf transplanting).The results showed that increasing the aperture of nursery site and adding microbial agent in the substrate to cultivate long-leaf tobacco seedlings could increase dry matter accumulation,improve dry matter distribution,increase nitrogen,phosphorus and potassium accumulation,promote fertilizer absorption efficiency,and increase fertilizer utilization rate and production efficiency;the contribution rates of aperture of nursery site,microbial agent,transplanting leaf age and the interaction of three factors to dry matter accumulation were 22.87%,27.73%,37.41% and 11.99%,respectively.The contribution rates to nitrogen accumulation were 20.34%,23.45%,33.62% and 22.59%,respectively.The contribution rates of phosphorus accumulation were 24.41%,27.81%,32.85% and 14.93%,respectively.The contribution rates of potassium accumulation were 15.48%,26.05%,34.61% and 23.86%,respectively.The contribution rates to nitrogen efficiency were 20.81%,44.67%,23.11% and 11.41%,respectively.The contribution rates of phosphorus fertilizer efficiency were 32.15%,31.66%,14.91% and 21.28%,respectively.The contribution rates of potassium fertilizer efficiency were 25.31%,38.71%,31.67% and 4.31%,respectively.In Hunan paddy-tobacco growing area,increasing the aperture of nursery site and adding microbial agents to the substrate can make up for the defects of two-stage seedling raising,and jointly improve nutrient uptake and fertilizer utilization efficiency,which has a certain popularization value in flue-cured tobacco production.

In order to understand the application effect of rice-straw application combined with decomposing agent on tobacco.The pot culture experiment was conducted to analyze the variation of the tobacco's growth,yield,chemical composition and coordination after the rice-straw application combined with chemical fertilizer and decaying agent containing different microbial dominant strains respectively,the research object was to explore the effects of rice-straw application combined with decomposing agent on the tobacco's growth,yield and quality.The results showed that the rice-straw application combined with decaying agent significantly increased the stem circumference,maximum leaf area,biomass,yields and the ratio of medium & superior grade of tobacco.The rice-straw application combined with decaying agent significantly lowered nicotine content of the upper tobacco,and increased its' total sugar content,reducing sugar content and total sugar-nicotine ratio,which resulted in reducing the irritability,bitterness and pungency of the upper tobacco and improving its' smoking quality;meanwhile,the rice-straw application combined with decaying agent significantly raised the content of total nitrogen,total sugar and reducing sugar in central tobacco,but there were no obvious variations for the ratio of total nitrogen to nicotine,reducing sugar total nitrogen and reducing sugar to nicotine,which meant the effect of rice-straw application combined with decaying agent on the central tobacco's chemical coordination was not significant;moreover,the rice-straw application combined with decaying agent increased the content of total sugar and reduced sugar in lower tobacco,and raised its' ratio of reducing sugar to total nitrogen and sugar to nicotine,which was beneficial to increase the aroma of lower tobacco and reduce its' impurity.According to the results of principal component regions between tobacco's agronomic characters and biomass,chemical composition and coordination indexes of different parts of cured tobacco,the rice-straw application combined with the decomposing agent containing dominant microorganism of bacteria+fungi or bacteria+fungi+actinomycetes had no significant difference in promoting tobacco's growth,yield and quality.Therefore,the rice-straw application combined with the decomposing agent containing different types of dominant microorganism could promote tobacco's growth,rationalize the chemical composition of upper and lower tobacco,and be beneficial to improve its sensory quality and increase tobacco's yield and quality.

To explore the effect of planting density on disease and quality of tobacco and the response characteristics of soil microorganisms in rainy regions,a field experiment was conducted in the Rainy Regions.The random block design was adopted to study the effects of different planting densities(CK was 16 650 and T₁ was 18 525 plants/ha) on yield of tobacco and soil microbial community structure during the experimental periods. Results suggested that:compared with the CK treatment,T1 treatment increased by 6.02% for the tobacco plant height,and 8.65% for yield,respectively. Compared with CK treatment,T1 treatment decreased by 15.38% for tobacco black shank,11.76% for tobacco black root rot,and 18.18% for tobacco anthracnose at the mature stage,respectively. T1 treatment increased by 1.62%for the species diversity and decreased by 0.99% for the richness of the rhizosphere soil bacterial community,increased by 3.76% for the species richness and decreased by 4.71% for the diversity of the fungal community when compared with CK treatment. The bacterial and fungal community structure of the T1 treatment was similar to that of the CK treatment,but the microbial dominant taxa in the soil samples was inconsistent. In the horizontal structure of phylum,T1 treatment relative abundance of bacteria in the soil Proteobacter,Acidobacteria and Actinobacteria,and fungal in the soil Ascomycota was higher. In the horizontal structure of genera,T1 treatment relative abundance of bacteria Sphingomonas and fungi Penicillium was significantly improved. Redundancy analysis (RDA)showed that environmental factors (sunshine duration,rainfall,and temperature)significantly affected the bacterial and fungal community structure,and the influence of soil bacterial community was rainfall>temperature>sunshine duration,the influence of soil fungal community was sunshine duration>rainfall>temperature,respectively. This study indicated that properly increasing the planting density of flue-cured tobacco in rainy regions could improve the soil microenvironment,and reduce tobacco diseases,which could improve the quality of flue-cured tobacco and increase the income of tobacco farmers.

In order to study the biological function of NbEHD1 in Nicotiana benthamiana,its gene structure,conserved domains,phosphorylation sites,subcellular localization and evolutionary relationship were predicted by bioinformatics methods.The encoding sequence of NbEHD1 was 1 638 bp,and its genomic sequence contained 16 exons and 15 introns.NbEHD1 protein was predicted located in the cytoplasm,which had 42 phosphorylation sites and had no signal peptide and transmembrane region.NbEHD1 belonged to the P-loop_NTPase superfamily and had a conserved domain specific to the EHD family.Phylogenetic relationship showed that NbEHD1 was closely related to EHD sequences of Solanum tuberosum and Solanum lycopersicum.Blast analysis against N.benthamiana sub-data base in SNG was performed to obtain the predicted full length of NbEHD1,then its full nucleic acid sequence was amplified by gene-specific primers.After obtaining NbEHD1 CRISPR/Cas9 gene editing construct,the vector was successfully transformed into the leaves of N.benthamiana by Agrobacterium-mediated genetic transformation.Eventually 16 T₀-generation plantlets were successfully identified,which provided materials for further research to determine the biological function of NbEHD1.

To investigate the effects of different maize straw carbon addition on soil organic carbon and fertilizer improvement,it focused on an in-situ soil column incubation experiment was established on tobacco fields.Four treatments were set,including none straw addition(CK),conventional straw(RS),decomposed straw(DS)and straw biochar(BC).The organic carbon components and structure,soil physical and chemical properties,and enzyme activities of the 0-20 cm soil layer of tobacco field treaded by the four treatments were characterized and analyzed.One-year incubation results showed that:Compared with none straw addition,the three forms of straw carbon addition all significantly increased the content of total organic carbon(TOC)and microbial biomass carbon(MBC)in the soil.Compared with none straw addition,treatments of conventional straw and decomposed straw significantly increased soil dissolved organic carbon(DOC)content,while straw biochar treatment significantly increased particulate organic carbon(POC)content(42.40%).The three forms of straw carbon addition all significantly increased the microbial biomass carbon/total organic carbon ratio and promoted the turnover of soil organic carbon.The functional group structure of SOC was analyzed by ¹³C nuclear magnetic resonance.The results indicated that conventional straw and decomposed straw treatment increased the relative content of Alkoxy carbon(O-Alkyl-C)and Carbonyl carbon(Carbonyl-C)(labile carbon component),while straw biochar treatment increased Alkyl carbon(Alkyl-C)(recalcitrant carbon component),Alkyl carbon/Alkoxy carbon(A/OA)and Hydrophobic-C/Hydrophilic-C(Hydrophobic-C/Hydrophilic-C).Different forms of straw carbon had significantly different effects on soil nutrients and enzyme activities,straw biochar treatment significantly increased by 65.72%,19.93% and 5.77% for the content of available potassium(AK),ammonium nitrogen(AN)and nitrate nitrogen(NN),respectively.Conventional straw and straw biochar treatments significantly increased the activities of sucrase(Su)and urease(Ur).The three forms of straw carbon all had a significant effect on the activity of phosphatase(Ps).Redundancy analysis showed that C/N ratio(C/N),cellulase(Ce)and total nitrogen(TN)were the main factors affecting the organic carbon content,while cellulase,pH,and available potassium were the main factors affecting the organic carbon structure.Overall,the application of maize straw biochar had significant advantages in improving organic carbon content and stability,nutrient content,and enzyme activity in a short term.In the comprehensive soil fertility evaluation,straw biochar had the highest score with 0.57.Maize straw biochar was an effective measure to improve the organic carbon level and soil fertility in the tobacco planting soil.

In order to investigate the growth, dry matter accumulation and nutrient use efficiency of rich flavor style flue-cured tobacco in Hunan paddy-tobacco, case study methods were carried out in two typical paddy-tobacco fields, suitable yield level(2 250-2 700 kg/ha) and high yield level(2 700-3 150 kg/ha), in Chenzhou Guiyang County and Hengyang Leiyang of Hunan Province in 2019. The differences in agronomic traits, leaf area index, root morphology, dry matter accumulation and distribution, nutrient accumulation and distribution, and nutrient utilization efficiency of flue-cured tobacco in different periods were analyzed. The results showed that:The above-ground part of flue-cured tobacco grown well at high yield level, the root system was well developed, the dry matter and N, P, K nutrients accumulated much, the utilization efficiency of N, P, K fertilizer was high, but the proportion of N, P, K nutrients in flue-cured tobacco leaves at suitable yield level was large, N, P, K nutrient harvest index was high. Flue-cured tobacco plants in Leiyang were tall, thick stems, thick roots, the dry matter and nutrient accumulation in roots and stems were higher;The leaf area of flue-cured tobacco in Guiyang County was large, many lateral roots, and the dry matter and nutrition of flue-cured tobacco were more accumulated. In the production of flue-cured tobacco of rice-tobacco rotation, in view of large soil masses and low temperature and rainy weather, it is necessary to mature the soil, cultivate strong seedlings, improve the quality of transplanting, create a good soil environment for the root growth of flue-cured tobacco, and promote the early growth and rapid development of flue-cured tobacco.

In order to improve the soil environment and soil quality of tobacco planting in rice growing area of Changsha. Through field experiments, 1 deep vertical rotary tillage(T1) treatment and 2 conventional controls were set up, including the ploughed furrow(T2) and direct rotary ploughing(T3), to explore the effects of different tillage methods on soil physical characteristics and dry and fresh weight of flue-cured tobacco roots in the rice-tobacco zone area in Changsha. The results showed that deep vertical rotary tillage improved the soil surface physical structure. Within the overall range of 0-30 cm surface layer, T1 deep vertical rotary tillage treatment showed an average reduction in bulk density and compactness of 8.8%, 29.7%, and an average increase in porosity of 10.67 percentage points in 2018, compared with T3 conventional treatment(local tillage method). In 2019, T1's bulk density and compactness decreased by 11.25%, 21.45% on average compared with T3, and porosity increased by 12.8 percentage on average. Within 3-5 months of flue-cured tobacco transplanting, the average moisture content of the soil at T1 was 0.62 percentage points higher than that at T2 in 0-10 cm, 3.23, 4.92 percentage points higher than that at T2 and T3 in 10-20 cm, 9.43, 12.3 percentage points higher at 20-30 cm, 9.56, 13.88 percentage points higher at 30-40 cm, respectively. At the same time, compared with conventional treatment, the D value was increased and the stability of soil aggregates was improved. In addition, the dry mass of flue-cured tobacco roots under T1 treatment was 69.75%, 28.93% and 28.33% higher at maturity than that under T3 treatment, and the volume of roots was 20.07%, 55.61% and 36.34% higher than that under T3 treatment. To sum up, deep vertical rotary tillage bed improved soil physical properties such as soil bulk density, compactness, porosity, soil moisture, soil aggregate stability and so on, improved soil environment, increased the biomass and volume of flue-cured tobacco roots and laid a foundation for improving the yield and output value of tobacco leaves.

To investigate the response law of organic carbon components and carbon conversion enzyme activities under different fertilization models, this study focused on long-term soil fertility tests that began in 2009, selected no fertilizer(CK), fertilization of chemical fertilizer(T1), organic manure with chemical fertilizer(T2) and fertilization of organic manure(T3) four trial processing.Compared with CK, T1 treatments, T2 and T3 treatments significantly improved TOC, DOC, MBC, ROC, CPMI and S-CL, S-SC, S-β-GC, S-PPD and S-POD.Pearson correlation analysis showed that DOC, MBC, ROC and S-CL, S-SC, S-β-GC, S-PPD and S-POD were extremely significantly positively correlated(P<0.01);Path analysis showed that S-CL, S-SC and S-β-GC directly affected the content of DOC, MBC and ROC, while S-PPD and S-POD mainly played an indirect role.Principal component analysis showed that the comprehensive effected of different fertilization models on active organic carbon components and enzyme activity could be divided into two groups:CK and T1 treatments as a group, T2 and T3 treatments as a group;combined with principal component analysis comprehensive score and analysis of variance, the order of different fertilization measured affecting soil active organic carbon components and enzyme activities was T3>T2>T1>CK. The application of organic fertilizer was the most effective fertilization measure to enhance the active organic carbon component and enzyme activity of tobacco-growing brown soil for Shandong smoke zone.

To determine the genetic diversity and genetic structure of Phytophthora nicotianae from Henan Province,34 P. nicotianae isolates were analyzed by randomly amplified polymorphic DNA (RAPD) marker. The results showed that 30 bands were amplified using 6 RAPD primers and 90% of them were polymorphic. Nei's gene diversity index was 0.271 0,and Shannon's information index was 0.402 8. The genetic similarity coefficient of 34 isolates ranged from 0.59 to 1.00. Based on UPGMA dendrogram,34 isolates were divided into 6 groups with 0.80 genetic similarity. Group Ⅰ was the dominant group including 26 isolates. The result of principle coordinate analysis was basically similar to that of UPGMA dendrogram. The result of genetic structure analysis revealed that P.nicotianae population in Henan originated from 2 ancestral subpopulations. The subpopulation Ⅰ and subpopulation Ⅱ accounted for 72.73%,27.27%,respectively. The subpopulation Ⅰ was the dominant subpopulation. Genetic component of 55.88% of isolates almost consisted of only one subpopulation. Therefore,the genetic diversity of P.nicotianae from Henan was low and the genetic structure of them was also simple.

In order to study the effect of different rootstocks on the disease resistance of tobacco grafted seedlings, and to clarify the mechanism of Yunyan 87 (Y87) as rootstock to improve the resistance of scion Hongdajinyuan (HD) to bacterial wilt, this study used split grafting and raw material band winding method to construct HD/HD grafted and HD/Y87 flue-cured tobacco seedlings respectively, using HD as the scion and Y87 as the rootstock. The PAL activity of HD/HD and HD/Y87 protective enzymes was measured. Finally, mRNA of 5 HD/HD or HD/Y87 were extracted and reverse transcribed to construct a total cDNA library by high-throughput sequencing technology and Illumina HiSeqTM 4000 sequencing platform. After sequencing and filtering low-quality Reads, the reference genome was compared with TopHat2, and the genes expression amount and differentially expressed genes were obtained by Cufflinks and DESeq software Heterogene expression. The results showed that the PAL enzyme activity of HD/Y87 was significantly higher than that of HD/HD at different sampling times, 48 414 474-48 697 874 Clean Reads and 38 272-40 938 genes were obtained from four samples, respectively, and the number of selective shear events of HD/Y87 was higher than HD/HD. After comparing the gene expression between HD/Y87 and HD/HD, 3 904 differential genes were obtained, 3 096 genes were up-regulated, 808 genes were down regulated; among the up-regulated genes, there were 3 genes ecoding phenylalanine ammonia lyases, 5 genes ecoding Myb family transcription factors, and 5 genes ecoding polyphenol oxidase involved in lignin synthesis, it also had 2 disease-related proteins and 3 genes ecoding ERF transcription factors. These results showed that Y87 as rootstock could improve the activity of HD PAL and gene expression of various resistance genes (including PAL, PPO and disease related protein) in scion, so as to improve the resistance of HD to bacterial wilt and other diseases.

A plant expression vector pBI121 -StPR1 was constructed to clarify the role of potato StPR1 gene in disease resistance, and introduce StPR1 gene into tobacco by Agrobacterium transformation method. The disease resistance and physiological characteristics of transgenic tobacco were investigated using different pathogen treatment, including bacterial disease soft rot (E. carotovora subsp. Carotovora Borgey, Ecc; E. chrysanthemi Burkholder. Atroseptica Dye, Ech; E. carotovora subsp. Mc Fadden et Dimock, Eca), bacterial wilt (Ralstonia solanacearum, RS) and fungal disease dry rot (F. sambucinum, F. avenaceum). The results showed that the diameter of lesions leaves increased with the prolongation of stress time in transgenic tobacco and wild type (WT) under the pathogen stress, and the diameter of transgenic tobacco lesions were significantly smaller than WT. In the physiological characteristics analysis, the physiological indexes of leaves increased with the prolongation of stress time in WT and transgenic tobacco, but the activities of SOD, POD and CAT in transgenic tobacco were all increased to different degrees compared WT. The results of disease resistance and physiological characteristics showed that transgenic tobacco had stronger resistance to pathogenic bacteria and fungi, indicating that the StPR1 gene played an important role in the disease resistance of potato, which could provide a theoretical basis for PR1 gene resistance in plants.

In order to explore the function of BdDREB1-like gene in transgenic tobacco, it was cloned and analyzed through bioinformatics technology. The open reading frame of this gene was 684 bp, encoded 228 amino acids, and the molecular weight of its encoding protein was 24.178 ku. It contained only one typical AP2 domain and belonged to the DREB family of AP2/EREBP superfamily. Multiple sequence alignment and evolutionary analysis showed that it was closely related to Rye ScCBFI. Subcellular localization analysis showed that BdDREB1-like was located in the nucleus. It was then constructed into plant expression vector and transformed into tobacco by Agrobacterium-mediated method to further confirm its function. PCR and RT-PCR screening showed that BdDREB1-like gene was not only integrated into tobacco genome, but also expressed stably. Transgenic tobacco seedlings with BdDREB1-like gene had higher germination rate under hydrogen peroxide stress. In order to further determine the relationship between BdDREB1-like gene and oxidative stress, we treated the one-month-old transgenic tobacco plants under oxidative stress with MV (Methyl viologen). The results showed that there were no significant differences in the contents of hydrogen peroxide, catalase and other physiological indicators except for SOD between wild type and T3 transgenic line, while there were only two physiological indexes of POD and chloroplast content of T1 transgenic line, which were significantly different from those of wild type under normal growth condition. Under oxidative stress, transgenic tobacco plants with BdDREB1-like gene had lower malondialdehyde and hydrogen peroxide content, higher peroxidase activity, catalase, superoxide dismutase and chlorophyll content compared with wild type. These results suggested that overexpression of BdDREB1-like gene enhanced the tolerance of plant to oxidative stress.

In order to explore the effects of tillage depth on the growth, development, yield and quality of tobacco plants in the Zhaoyang tobacco-growing area, there was experimental research on the depth of the tillage to the treatments 20 cm (T1), 25 cm (T2), 30 cm (T3) and CK (15 cm) by the randomized block design in the field. The results showed that there were significantly different at 0.05 of volume-weight and soil porosity between T2 and T1 in the long-term, and there was significantly different at 0.05 level of volume-weight between T2 and T3. The T2 dry matter accumulation of tobacco plants roots, stems and leaves was comparatively large at different times, followed by T3 and T1, and CK was the worst. The root-knot nematode disease of T3 was the lightest, the incidence rate was 25.23%, and there was significantly different at 0.05 level with CK; the disease index T3 (4.04) < T1 (5.27) < T2 (5.39), significantly lower than CK (18.64) at 0.01 level. In the agronomic traits, the plant height of T2 was 112.85 cm, the leaf area coefficient of T2 was 3.09, and the stem circumference of T3 was 12.07 cm, which was significantly higher than CK at 0.05 level. In the economic traits, T2 yield, upper grade tobacco ratio (α=0.01), medium grade tobacco ratio (α=0.05) were significantly higher than CK; T3 yield (α=0.05) was significantly higher than CK; T1 medium grade tobacco ratio (α=0.01) was significantly higher than CK. On the whole, the economic traits of T2 flue-cured tobacco was the best, followed by T3 and T1, and CK was the worst. The study found that the continuous ploughing depth of 25-30 cm can improve soil physical properties, reduce the occurrence of root-knot nematode disease, promote the growth and development of tobacco plants, and improve the yield and quality of tobacco.

The aims of this study were to reveal the appropriate mode for water-potassium coupling of flue-cured tobacco in Qiandongnan Prefecture, Guizhou province. Three irrigation rates: 0.50 L/plant(W₁), 1.00 L/plant(W₁) and 2.00 L/plant(W₃), and three potassium fertilizer rates:135 kg/ha(K₁), 270 kg/ha(K₂) and 405 kg/ha(K₃) were set in this field plot experiment, resulting in a total of nine treatments. The effects of different potassium application rate and water irrigation rate coupling treatments on the growth of flue-cured tobacco, yield and quality of tobacco leaf were studied. Different water and fertilizer amounts significantly or extremely significant influenced the plant height, maximum leaf area, stem girth and root volume, and interaction effect existed between water and fertilizer amounts. Water and fertilizer amounts significantly influenced total sugar, reduced sugar, alkaloid, total nitrogen, potassium and chloride contents in tobacco leaves(C3F grade), the smoking quality and appearance quality of flue-cured tobacco leaf increased with the increase of irrigation rate and K rate, and interaction effect existed between them.Water and fertilizer rate significantly influenced tobacco yield, and interaction effect existed between them. Considering all above, when irrigation water quantity was 1.00-2.00 L/plant, K application rate was 270 kg/ha (W₂K₂ and W₃K₂), the growth of tobacco plant, yield and quality of tobacco leaves were improved. Linear fitting revealed a positive relationship between plant height, root volume and yield of tobacco leaves within a certain range. When irrigation rate was 1.00-2.00 L per plant, K application rate was 270 kg/ha, water-potassium coupling treatment had a good effect in promoting tobacco plant growth, increasing yield and improving quality of tobacco leaves in tobacco-planting in Qiandongnan Prefecture, Guizhou province. From the view of obtaining high yield and saving water and fertilizer, W₂K₂(1.00 L/plant, 270 kg/ha)and W₃K₂(2.00 L/plant, 270 kg/ha)treatment can be used as an appropriate combination under the present experimental conditions.

In order to clarify the decomposing characteristics of different rates of tobacco straw and their effects on rice yield. In this study, one field experiment was conducted. Then, the decomposition rates of tobacco straw and rice yield among different treatments were analyzed. Meanwhile, the quantitative relationship between the external input of carbon and nitrogen ratio and rice yield was discussed. The results showed that： the decomposing characteristics of the tobacco straw in all treatments were fast in the early stage and slow in the late stage. At the 5th day, the amount of decomposed tobacco and the average daily decomposing amount were reached the maximum, which were 0.87 g and 0.29 g/d, respectively. The cumulative decomposing rate of tobacco straw also showed a similar trend, and the cumulative decomposing rate of tobacco straw in the whole rice season was 42.31%. However, there was not significant different for the total and daily decomposed amount of tobacco straw among all treatments, but the cumulative decomposing rate of the semi-returning treatment was higher than the equivalent and 1.5 times returning. The correlation between the input of carbon-nitrogen ratio and rice yield could be fitted by the quadratic curve equation. Moreover, it indicated that the reasonable input of carbon-nitrogen ratio was 10.1, and the corresponding maximum yield of rice was 10.4 t/ha. Therefore, in the rotation of tobacco and rice, the reasonable carbon-nitrogen ratio input was one key pattern to improve rice yield, although the characteristics of decomposing under different amounts of tobacco straw was similar.

In order to discover the function of Blind gene in Nicotiana tabacum, we cloned one Blind-like gene, NtBL1, from K326 by homologue cloning method. The protein domain and structure was predicted by PROSITE(ExPASy), Sopma and Swissmodel, the Phylogeny tree construction and Motif prediction of NtBL1 and its homologues were analyzed separately by Mega 7.0 and MEME, the expression pattern of NtBL1 in different tissues was analyzed by semi-quantitative PCR, and the expression changes of NtBL1 under abiotic stress treatment was detected by qRT-PCR. The results showed that the coding sequence of NtBL1 was 1 014 bp, encoding 337 aa. The expression pattern of NtBL1 in transcription level was generally detected in several organs and growing stages, while it had the highest expression level in root. Bioinformation analysis showed that NtBL1 was a typical R2R3 Myb protein, which contained 2 Myb-type HTH domains. The phylogenetic tree analysis showed that NtBL1 gene was shared lots of motifs with tomato Blind. The results of qRT-PCR showed that the expression level of NtBL1 gene was up-regulated by ABA treatment and the highest expression level appeared in 24 h treatment; The expression level of NtBL1 gene was down-regulated by NaCl treatment and the lowest expression level presented in 24 h treatment. In conclusion, NtBL1 gene might participate in regulation of axillary mersitem development by ABA and NaCl.

In order to further clarify the mechanism of over expression TaZAT8, a wheat C2H2 type zinc finger gene, to enhance the resistance of tobacco plants under P-starvation, the classical proteomics approach was applied to reveal the changes of protein expression between WT and transgenic tobacco plants. The results showed that there were 22 protein spots differentially expressed on 2-DE images, and among them, twenty protein spots were up-regulated and two were down-regulated respectively. Subsequently, these protein spots were identified by LC-MS/MS, which were classified into six functional groups, including primary metabolism, energy generation, stress response and defense, transcription and translation, protein turnover and unclear functions. Subcellular localization analysis indicated that these proteins were mainly located in five organelles, including cytoplasm, mitochondria, peroxisome, nucleus and chloroplast. Go analysis showed that the differentially expressed proteins participated in various metabolism processes or act as enzymes of different categories. Above results showed that over expression TaZAT8 laid the foundation for improving the plant tolerance to stress via regulating proteins involved in biological processes such as substance metabolism, energy production, stress response, etc.

The effects of green manure intercropping on the functional diversity of soil microbial communities in tobacco growing soils were studied by Biolog technique to provide the theoretical basis for application of green manure intercropping with tobacco.The average well color development (AWCD) of soil microbial carbon metabolic activity at the stages of rosette and maturity were intercropped with Melilotus officinali > Lolium > control, indicating that intercropping with green manure had a significant effect on soil microbial carbon utilization. The Shannon index,Simpson index and Mclntosh index of intercropping green manure were higher than those of control, while the soil microbial Shannon index, Simpson index and Mclntosh index of intercropping Melilotus officinalis had the highest value. The results showed that intercropping green manure affected the microbial community structure of tobacco growing soil and improved the microbial community diversity of tobacco growing soil. Metabolic intensity analysis of different carbon sources showed that the utilization of amino acid, carboxylic acid and carbohydrate by soil microorganisms in different green manure and different growth stages was significantly higher than that of ammonia/amine, miscellaneou and polymer carbon sources. Principal component analysis showed that the functional diversity of soil microbial community was significantly different, and carboxylic acids, amino acids and sugars were the three main carbon sources, which led to the difference in soil microbial metabolism. Intercropping green manure enhances the ability of soil microorganism to utilize carbon source, and improves the diversity of soil microbial community structure and community function in tobacco growing soils. The effect of intercropping Melilotus officinalis is more obvious.

In order to set up the basic theory for rational fertilization technology of tobacco in Southern Anhui Province,we analyzed the changes of agronomic characters,dry matter accumulation,nutrient absorption and fertilizer use efficiency of flue-cured tobacco under different nitrogen and potassium fertilizer application rates. The result showed that high nitrogen fertilizer rate caused the increment of plant height,leaf number,leaf length and leaf width of tobacco at different positions. The dry matter accumulation in different organs and the ratio of shoot to root also increased with the increase of nitrogen application rate. The increase of nitrogen fertilizer rate also increased the nitrogen and potassium accumulation in roots,stems and leaves. The amount of nitrogen fertilizer was positively correlated with the whole plant nitrogen concentration,and had significantly positive correlation with the total nitrogen and potassium accumulation. With the increase of nitrogen fertilizer amount,the utilization rate of nitrogen fertilizer did not change significantly,but the agronomic efficiency and partial productivity of nitrogen fertilizer increased significantly. The increase of potassium fertilizer had no significant effect on the agronomic traits of flue-cured tobacco. High potassium fertilizer rate caused the increment of nitrogen concentration in roots,and potassium concentration in stems and leaves,but the dry matter accumulation and nutrition accumulation in different organs were not influenced by potassium fertilizer rate. The amount of potassium fertilizer was only positively correlated with the whole plant potassium concentration. The utilization rate of potassium fertilizer was relatively low among the treatments. Among the treatments with the lowest application rate of potassium fertilizer,the utilization rate of potassium fertilizer,the agronomic efficiency and partial productivity of potassium fertilizer were the highest. In the experiment,the dry matter accumulation and the nutrition accumulation in whole plant,and the nitrogen fertilizer use efficiency increased significantly with the increment of nitrogen application rate. Applying nitrogen fertilizer had obvious effect on the increase of yield. With the increase of potassium fertilizer,there was no significant change in dry matter accumulation and nutrient accumulation. According to the results of leaf potassium concentration and the potassium fertilizer use efficiency,the potassium application rate should be reduced in Southern Anhui Province.

The aim of this study was to reveal the effects of application rates of chemical nitrogen(N) fertilizer combined with organic fertilizer(rapeseed cake,M)on the quality,N uptake and use efficiency of tobacco Yun yan 87,and to find the best organic and inorganic N application amount of tobacco in Qiandongnan prefecture of Guizhou Province,which could provide the technical reference for reducing the production cost,and increasing the yield and quality of tobacco. Six treatments,i.e. CK₁,CK₂ and MN_iPK(i=0,1,2 or 3)were set in a field plot experiment. The effects of each treatment on the leaf quality,N uptake and use efficiency of tobacco were analyzed. The results showed that,compared with CK₂ treatment,total sugar contents in tobacco leaves of MN₁PK,MN₂PK and MN₃PK treatments increased by 8.57%,14.18% and 13.03%,respectively,the reduced-sugar contents increased by 4.67%,9.66% and 8.26%,respectively,the potassium contents increased by 8.34%,19.08% and 17.12%,respectively,while the chlorine content decreased by 0.36%,8.19% and 3.56%,respectively. The ratio of potassium to chloride and the difference between total sugar and reduced-sugar were improved. Compared with CK₂ treatment,the stimulation and foreign smoke of tobacco leaves decreased and the fragrance quality and fragrance amount enhanced in combination of chemical N fertilizer with organic fertilizer. The combustibility enhanced,and the lasting smoke was more comfortable and longer. Compared with MN₁PK and MN₃PK treatments,MN₂PK treatment increased the N use efficiency by 2.99,4.54 percentage points,respectively. The chemical N fertilizer combined with organic fertilizer(organic N accounting for 36.00%-45.77%)coordinated the ratio of different chemical component of tobacco leaves,improved the smoking quality,increased N the nitrogen uptake of tobacco plant and enhanced N use efficiency. After comprehensively analyzing the effects of each treatment on chemical component,smoking quality and N use efficiency of tobacco leaves,the best treatment was that the organic N accounted for 45.77%,and the recommended ratio of organic N was 36.00%-45.77% of total N in tobacco land of Qiandongnan prefecture,which was suggested to be extended in the local areas,and could provide a reference for the neighboring areas.

It is known that the threonine at position 1 084 of the third coiled-coil (CC3) of the new member of the tobacco-driven family NtTkr is crucial for the binding of the target protein. Multiple candidate proteins interacting with NtTkr were obtained by yeast two-hybrid screening of NtTkr tail. In order to determine the importance of T₁₀₈₄ deletion or replacement mutation between NtTkr and target protein vitro, first the pBI121-NtTkr plasmid was take as a template to obtain the tobacco NtTkr tail T₁₀₈₄ deletion and replacement tail by overlap extension PCR, and cloned T_1084d,T1084A into pUC19, by the blue white spot screening, Sma Ⅰ-BamH Ⅰ double enzyme cuting the identification and gene sequencing, geting the right T₁₀₈₄ deletion and replacement NtTkr tail; Then restructured pUC19-T_1084d, pUC19-T1084A and pMXB10 with Not Ⅰ-Nde Ⅰ double enzyme, the target fragment and the carrier fragment are recovered and connected, and the ligation product transforms DH5α. The recombinant was screened to Not Ⅰ-Nde Ⅰ double enzyme identification, managed to build the required pMXB10-NtTkr-T1084A and pMXB10-NtTkr-T_1084d prokaryotic expression vector; Finally, pMXB10-NtTkr-T1084A and pMXB10 -NtTkr-T_1084dd were transferred into BL21 (DE3), and the protein expression was detected by 12% SDS-PAGE after the induction of 0.05 and 0.06 mmol/L IPTG concentrations, respectively NtTkr-T1084A-1320 of about 77 ku and NtTkr -T_1084d-1317 of 76.2 ku were highly expressed.

Under the optimization of C/N with organic fertilizer and N fertilizer, a field experiment was carried out to study the effect of potassium and the ratio of basic/dressing fertilizer on biomass, potassium uptake, soil available potassium content and aroma quality of tobacco. Split-plot experiment design was adopted. The main treatment was 3 K level:K1 (120.0 kg/ha),K2 (240.0 kg/ha) and K3 (360.0 kg/ha);and the subsidiary treatment was the ratio of basic/dressing K fertilizer:S1(7:3),S2(5:5) and S3(3:7). The results showed that the biomass of tobacco leaves increased with the increase of potassium application, while it decreased with the increase of potassium dressing ratio in the same K level in the fast growth and mature period. Under the K2 and K3 level, the leaf potassium content was lower in the S1 treatment than in S2 and S3 treatments, indicating that the increase of K dressing percentage was conducive to the transfer of potassium ions from lower leaves to middle and upper leaves. With the increase of potassium application, the content of available and slowly available potassium in rhizosphere soil increased. Under K2 level, the available potassium of soil reached to maximum at fast growth and bud stages, and was lower in S3 treatment than in S1 and S2 treatments at mature stage. Under K3 level, the available potassium in rhizosphere soil had no significant difference among the four growth stages, in which that of S3 treatment > S1 treatment > S2 treatment. Compared with other treatments, the contents of total nitrogen, total nicotine and protein in tobacco leaves were significantly higher in S3 treatment under K2 level, and the ratio of sugar/alkali and ratio of nitrogen/alkali were improved. The neutral aroma components had no significant difference in upper leaves under K1 level, but it improved significantly in S3 treatment under K2 and K3 levels, especially for S3 treatment under the K2 level, in which the quality, quantity and concentration of aroma in tobacco leaves were the best. It was concluded that under the optimization conditions of organic fertilizer C/N, 240.0 kg/ha potassium fertilizer was recommended in the sand mud soil of Southern China, and the basic fertilizer was 72.0 kg/ha and the dressing one was 168.0 kg/ha (the ratio of base to dressing was 3:7), which could effectively improve the potassium content and quality of tobacco leaves.

In order to improve the quality of soil and tobacco,effects of precision fertilization of flue-cured tobacco on soil structure and quality and benefit of tobacco leaves were studied by way of field demonstration and plot experiment. The results demonstrated that the B2F total and reducing sugar were increased significantly by precision fertilization,while the B2F total vegetable alkali content decreased,the sugar alkali of flue-cured tobacco were adjusted,total soil porosity and the organic matter content and the capillary water holding capacity in the early and middle stage were improved,and smoking quality of B2F tobacco leaves were improved,such as strength,concentration,aroma quantity,aroma,aftertaste,and quality of B2F tobacco leaf was improved significantly,ratio of potassium to chlorine of C3F tobacco leaf was decreased. Along with the depth increase and reduction of fertilizer,precision fertilization was efficient between significantly positive correlation changed with the smoking quality of B2F tobacco leaf,precision fertilization 7cm+90% fertilizer could increased content of organic matter of soil,could significantly improved ratio of potassium to chlorine of B2F tobacco leaf,precision fertilization 12 cm deep fertilization could significantly increased ratio of potassium to chlorine of B2F tobacco leaf,significantly improved the quality of B2F tobacco leaves.Precision fertilization 7cm could improve the content of reducing sugar and total sugar of C3F tobacco leaf,and decreased the content of total vegetable alkali,could improved the smoking quality of tobacco leaves,such as strength,concentration,aroma quantity,aroma,aftertaste.

The effects of different phosphorus application rate on growth and yield of tobacco Yunyan 87 were studied to explore the suitable phosphorus application rate in Qiandongnan prefecture tobacco region, Guizhou province, and to provide scientific basis for phosphorus fertilizer application in tobacco production. A field plot experiment was conducted, which was established six fertilizer treatments, i.e. CK, M, P₀,P₁, P₂ and P₃. In the growth period of tobacco, main agronomic characters, diseases situation and yield of flue-cured tobacco were recorded and investigated. The results showed that P₃ treatment was the best treatment, which had good field growth, good agronomic characters, strong disease-resistant ability and high yield of the all fertilizer treatments. At mature stage, as compared with CK treatment, tobacco plant of P₃ treatment increased plant height, maximum leaf area, leaf numbers, stem girth, internodal length and root volume by 63.19%, 72.67%,78.93%,37.93%,13.65% and 161.79%, respectively; As compared with P₀ treatment, tobacco plant of P₃ treatment increased plant height, maximum leaf area, leaf numbers, stem girth and root volume by 15.90%,21.46%,23.61%,11.25% and 28.75%, respectively. As compared with CK and P₀ treatment, tobacco plant of P₃ treatment decreased incidence of Red star disease, Mosaic and Black shank by 7.51, 7.92, 6.68 and 3.66, 6.52, 4.72 percentage point, decreased disease index of Red star disease, Mosaic and Black shank by 56.12%, 45.53%, 51.33% and 33.72%, 31.97%,43.53%, respectively. As compared with P₂ and P₀ treatment, P₃ treatment increased tobacco leaf yield by120.62% and 28.51%, respectively. The suitable chemical phosphorus fertilizer application rate was 112.5 kg/ha in Qiandongnan prefecture tobacco land, Guizhou province. Therefore, it is suitable to be extended in this tobacco region and can provide a reference in its neighbouring areas.

The current agricultural cultivation is highly dependent on chemical fertilizers,resulting in the deterioration of the micro-ecological environment of the soil. In order to improve the biological characteristics of the soil and fertility, The effects of green manure (an annual) as green manure or chemical fertilizer on the activities of invertase,urease,phosphatase and catalase in flue-cured tobacco soil were studied. The results showed that the activity of four enzymes could be significantly increased by overturning of grass rhinoceros,and the soil enzyme activity increased with the increase of the amount of green manure. Compared with the blank control (CK2),the third batch of grass rhinoceros (overturned on September 18 with a biomass of 32 180 kg/ha) had the largest increase in enzyme activity,with the maximum increase being:sucrase June 48.4%,91.5% for urease in April,72.4% for phosphatase in August,and 51.6% for catalase in October. In the treatment of green manure and chemical fertilizers,the treatment of green manure + chemical fertilizer was higher than that of the control (CK1,only chemical fertilizer), the effect of the third batch of overturning green manure plus conventional fertilization on soil enzyme activity was better than that of green manure plus 30% nitrogen reduction, The first batch (turnover on August 3,biomass was 19 350 kg/ha) and the second batch (biomass,22 930 kg/ha on August 18) were overturned.The different of soil enzyme activity between green manure+conventional fertilization and green manure+nitrogen reduction 30% was not significant,and urease and phosphatase were particularly prominent. The characteristics of the dynamic changes of enzymes in different growth stages of tobacco plants were as follows:soil sucrase,urease,and phosphatase in all treatments with overturn green manure green manure,and chemical fertilizers all peaked in the long-term (June) peak. Catalase A peak appeared in the early stage (August) of tobacco leaf harvest. The above results indicated that the soil metabolism was very active after the rhizome was pressed,which was conducive to the nutrient transformation of the soil and provides the necessary nutrition for the normal growth of the flue-cured tobacco.

In order to make rational use of earthworm manure instead of some chemical fertilizer,improving the utilization efficiency of chemical fertilizer and soil quality in tobacco growing area of Southern Shaanxi.A field experiment with five treatments:CK (no fertilizer),T1 (chemical fertilizer),T2 (nitrogen fertilizer nitrogen replacement 30% earthworm manure),T3(nitrogen fertilizer nitrogen to replace 50% earthworm manure),T4(nitrogen fertilizer nitrogen replacement 70% earthworm manure).We studied the effects of different fertilization treatments on the physicochemical properties,enzyme activity and microorganism of tobacco planting soil,and analyzed the correlation of various fertility factors,and comprehensively evaluated the soil fertility level.The results showed that without fertilizing or applying earthworm manure,soil bulk density could be reduced significantly.Compared with pure fertilizer treatment (T1),no fertilizer treatment (CK) decreased 2.38%,and earthworm manure treatment (T2,T3 and T4) decreased 3.17%-6.35%.Fertilization could significantly increase soil available nutrients and organic matter content.The effect of T2 treatment on soil quick effective nutrients was the best,compared with CK treatment soil alkaline nitrogen,available phosphorus and available potassium,respectively,2.63,1.38 and 0.99 times more.The content of organic matter increased with the increased of the proportion of earthworm manure.The highest T4 content reached 22.35 g/kg,which was 62.07% higher than that of CK treatment.The replacement of some fertilizer by earthworm manure could significantly increase the biological characteristics of soil,and the activity of T1 urease,invertase,acid phosphatase and catalase increase 16.45%-37.97%,22.99%-34.57%,18.40%-28.80% and 10.19%-19.74%,respectively.While the number of bacteria,fungi and actinomycetes increased by 43.22%-122.35%,20.74%-55.87% and 44.80%-76.64% respectively.There was a certain correlation between the fertility factors.The principal component analysis showed that the first principal component reflected the transformation ability of soil organic matter,and the second principal component represented the supply capacity of soil available nutrients.The comprehensive model showed that the difference of the effect of earthworm feces partly replaced by inorganic fertilizer on soil comprehensive fertility was mainly reflected in available potassium,available phosphorus,alkali hydrolysable nitrogen,actinomycetes,fungi,acid phosphatase,bacteria and organic matter.Soil comprehensive fertility evaluation showed that T4 (earthworm manure nitrogen instead of 70% fertilizer nitrogen) was the best,and T2 (earthworm manure nitrogen instead of 30% fertilizer nitrogen) was the second.

In order to study the effect of garlic root decomposition on tobacco black shank in tobacco growing soil,the greenhouse pot experiment was carried out to study inhibition effects on tobacco black shank through indoor cultivation by garlic root decomposition with different decomposing time.The experimental result indicated that garlic root decomposition with different decomposing time could inhibit the growth of tobacco black shank fungus,and garlic root decomposition that decomposed 15 days had the best inhibition effect.Treated by garlic root decomposition that decomposed 15 days,myceliun appeared hyphae deformation,hyphae branch and protoplasm leak.Pot experiments showed that the control effect of garlic decomposition solutions decomposed in different times on tobacco black shank was to 4.30%-41.38% in ninth days after the transplanting of flue-cured tobacco.From the findings it is inferred that garlic root decomposition had a good inhibitory effect on tobacco black shank,and had highly activity on tobacco black shank.

The HKT protein family is mainly involved in controlling the absorption of K⁺ and the selective transport of K⁺/Na⁺, which plays an important role in improving the stress resistance of plants.In order to study the relationship between the tissue expression level of AtHKT1 and salt tolerance of plants,the homology of HKT proteins,the expression of AtHKT1 gene and the cis-acting elements of AtHKT1 promoter were analyzed and predicted using bioinformatics techniques. On the basis,the AtHKT1 promoter was introduced into Nicotiana benthamiana. GUS staining results were used to analyze the tissue expression level of AtHKT1 gene in Nicotiana benthamiana. The results of bioinformatics analysis showed that Arabidopsis thaliana and wheat were located in different evolutionary branches and their genetic relationship was far, suggesting that the function of AtHKT1 might be different from the corresponding protein in wheat. AtHKT1 gene was abundantly expressed in many organs and tissues of Arabidopsis thaliana, especially in leaves, roots and flowers, which confirmed that AtHKT1 gene was likely to have important physiological functions. The AtHKT1 promoter might be a stress-responsive promoter containing a number of important elements that responded to environmental stress. Therefore, the expression of AtHKT1 gene was likely to be regulated by environmental stress. Results of GUS staining showed that the coloration of leaves,vascular system,roots and flowers of the seedlings with pHKT1-gus was deeper,further confirming that the expression level of AtHKT1 in these regions was high. The above results indicated that the regulation of AtHKT1 gene expression was conducive to the translocation of Na⁺,thereby regulating the salt tolerance of plants.In addition, there might be other unknown features that further increase the complexity of AtHKT1 functionality. At present, the mechanism of K⁺ and Na⁺ transport of HKT proteins is not clear. The expression level of AtHKT1 gene in Nicotiana benthamiana was analyzed to provide reference for further understanding the action mechanism of AtHKT1 gene.

A field experiment was conducted to study the effect of compound-organic-bio-bacteria-fertilizer or/and biochar on soil properties and dissolved soil N and C in the tobacco-growing soil.Seven treatments were set as:①no organic fertilizer (CK),②biochar(T1),③Compound-organic-bio-bacteria-fertilizer (COBBF, T2),④Bio-bacteria-biochar-fertilizer(T3),⑤50% COBBF+50% biochar(T4)⑥60% COBBF+40% biochar(T5),⑦40% COBBF+60% biochar(T6). The result showed the soil nutrient was significantly improved under treatments with organic fertilizer application.The ammonium nitrogen content under T2 treatment was significantly promoted after tobacco transplanted 60 d compared with other treatments.The nitrate nitrogen content under T5 treatment was improved in the whole growth stage. The soil available potassium content under T2 and the soil available, phosphorus under T5 treatments were increased after tobacco transplanted 60 d compared with other treatments.The DOC content and DOC/SOC were obviously promoted under T1 and T3 treatments compared with other treatments, the highest DOC/SOC ratio as 8.31 mg/g occurred under T3 treatment. The DON content under T2 treatment peaked as 57.09 mg/kg after tobacco transplanted 90 d, while which reached as 97.98 mg/kg under T5 treatment during tobacco transplanted 60 d; Aromaticity index (AI) was reduced under T3,T4,T5 and T6 treatments. The E2/E3 was increased during tobacco transplanted 90 d. In conclusion, in central area of Henan Province, it is suggested that the COBBF only and 60% COBBF+40% biochar application were the optimal practice to improve soil N content;while biochar only and Bio-bacteria-biochar-fertilizer application could improve the soil dissolved C.

In order to study the function of CIPK3 in abiotic stress response,a CIPK family gene NtCIPK3 was cloned from common tobacco K326 by homologous cloning. The gene contained a 1 272 bp ORF encoding 423 amino acid. Protein sequence analysis indicated that the protein contained a transmembrane domain and belonged to a hydrophilic membrane protein. Protein alignment analysis revealed that NtCIPK3 contained a highly conserved N-terminal kinase region,a junction region,and a C-terminal regulatory region,with the highest homology to wild tobacco CIPK3,reaching 99%.Subcellular localization predictions showed that the protein was mainly located in the nucleus and had a bipartite typing nuclear localization signal sequence. The expression characteristics of this gene were analyzed by qRT-PCR. Analysis of tissue expression revealed that the gene was expressed in the roots,stems,leaves and flowers of tobacco and had tissue-specific expression and the expression level in leaves was significantly higher than that in other tissues,and it was speculated that it might function mainly in leaves.Meanwhile,NtCIPK3 gene was induced by low potassium,high salt,drought,ABA,H₂O₂,and low temperature treatment. It is speculated that NtCIPK3 gene played an important role in tobacco abiotic stress response. And successfully constructed the pBI121-NtCIPK3 overexpression vector,which laid the foundation for the future research of the gene under abiotic stress.

To explore the relationship between tobacco NtCIPK family gene expression and the potassium content of tobacco, and to provide reference for the molecular mechanism of potassium absorption in tobacco. For common tobacco varieties K326, Guiyan 5 and Yun 87 three different potassium concentrations (6.0, 1.0,0.2 mmol/L) were cultured in solution and their dry weight and potassium content were measured. At the same time, 11 genes of the NtCIPK family were analyzed for different potassium concentrations using qRT-PCR. Induced expression pattern. With the increased of potassium concentration in the culture medium, the dry weight and potassium content of the tested plants showed an increasing trend, and there were significant differences between treatments. Among them, Yun 87 is treated with different potassium concentrations (1.0, 0.2 mmol/L). Dry weight and potassium content were the highest. The expression of 11 genes in the NtCIPK family was induced by the change of potassium concentration, and showed up-regulated expression with the decreased of K⁺ concentration; only the expression of NtCIPK9 in K326 and NtCIPK24 in Guiyan 5 showed a downward trend.The results of path analysis showed that the expression of NtCIPK3 and NtCIPK5 was significantly positively correlated with the potassium content of K326 and Guiyan 5, respectively, at the concentration of 0.2 mmol/L K⁺, while NtCIPK23 was treated at the concentration of 6.0 mmol/L K⁺. The expression had a significant negative correlation with the potassium content per plant of Yun 87. The potassium content of tobacco was closely related to the expression of NtCIPK gene, and the enhanced expression of NtCIPK gene contributes to the absorption and accumulation of potassium by tobacco.

Potassium channel is the main protein of plant uptake and translocation K⁺,and SKOR (Stelar K⁺ outwaed rectifier) belongs to Shaker channel family exogenous rectifier,which plays a key role in response to low potassium stress response in plants.In order to study the function and role of tobacco NtSKOR gene in abiotic stress response,one NtSKOR gene was cloned from Nicotiana tabacum K326 with homologous cloning strategy,gene expression patterns were analyzed by Real-time quantitative PCR,and their physicochemical properties,domains,phosphorylation sites and evolutionary relationships of proteins were predicted by bioinformatic analysis. Bioinformatics analysis showed that this gene contained 2 484 bp and encoded 827 amino acid. The predicted molecular weight was 94.75 ku and the isoelectric point (pI) was 6.52. The largest secondary structure element of this protein was α-helix,and the smallest was β-turn. NtSKOR contained six transmembrane domains(S1-S6),with three different kinase phosphorylation sites for serine,threonine and tyrosine. Phylogenetic analysis showed that the protein had 99% homology with Nicotiana sylvestris and 96% homology with Nicotiana tompentosiformis,thus it was named as NtSKOR. Expression patterns showed that the gene was expressed in roots,stems,leaves and flowers in mature stage,which had the highest expression in roots and the lowest expression level in flowers. Expression patterns under abiotic stress indicated the gene could respond rapidly to the adverse conditions of low potassium,high salt,drought,H₂O₂,ABA and 4℃. This suggests that NtSKOR gene plays an important regulatory role in tobacco abiotic stress,and the results provide a theoretical basis for further in-depth study of the NtSKOR function.

In order to study the function of tobacco NtRAP2-7 gene in abiotic stress response, NtRAP2-7 was cloned from K326 by homologous cloning. And the bioinformatics software was used to analyze the physicochemical properties, spatial structure and phylogenetic analysis of the encoded protein. Sequence analysis results showed that the gene contained 1 398 bp in length which encoded 465 amino acid residues and the predicted molecular weight of this protein was 51.16 ku. Based on 3-D modeling and secondary structure analysis, the protein consisted of three β-sheet and one α-helix and contained 58 phosphorylation sites. The subcellular localization prediction showed that the protein was mainly located in the nucleus and contained a monopartite nuclear location signal sequence. Phylogenetic analysis showed that NtRAP2-7 protein had the highest homology with Nicotiana sylvestris RAP2-7 protein sequence, which was 98%.The expression pattern analysis of the gene was carried out by qRT-PCR. Tissue expression analysis showed that the gene was expressed in the roots, stems, leaves and flowers, but had the highest expression level in roots. The expression levels of NtRAP2-7 was affected by abiotic stress treatments, indicating that the NtRAP2-7 was involved in the tobacco abiotic stress response. The pBI121-NtRAP2-7 overexpression vector was successfully constructed by double enzyme digestion, which laid the foundation for further study on the function of this gene in abiotic stress.

This study laid the theoretical foundation for the study of the function and structure of peroxidase genes in tobacco and cloned the cDNA of peroxidase gene NtPOD1 from tobacco cultivar K326 by homologous cloning and analyzed the bioinformatics. At the same time,qRT-PCR method was used to analyze the expression pattern of tissue and organ of this gene and its stress response. The results showed that the total length of cDNA was 981 bp,encoding 326 amino acid residues,the predicted molecular weight was 37.19 ku,and the isoelectric point was 8.89. Bioinformatics analysis showed that the protein was a hydrophilic protein containing four conserved disulfide bonds and two conserved calcium binding sites in the domain,which may be located in extracellular(including cell wall).The class Ⅲ secretion oxidase of the dependent peroxidase superfamily had high homology with Nicotuana attenuata POD42, Nicotiana sylvestris POD42,Solanum tuberosum POD42 and so on. The gene was expressed in tobacco roots,stems,leaves and flowers,with the highest expression in leaves and the lowest expression in flowers. Meanwhile, NtPOD1 expression was induced by high salt,drought,hypokalemia,ABA and H₂O₂. The results indicate that NtPOD1 belongs to tobacco peroxidase and may play a role in the response of tobacco to abiotic stress.

In order to improve soil physical properties and study the long-term effects of continuous application of biochar on soil physical properties of planting tobacco. Yunyan 87 was selected as testing material and a three years located experiment was designed to study the effects of continuous application of biochar on soil physical properties of planting tobacco. The results showed that continuous application of biochar could obviously affect the soil physical properties. Continuous application of biochar promoted the soil moisture content in each periods of flue-cured tobacco growth in 2015,the soil moisture content increased by 3.243-3.983 percentage points,which had extremely significant difference,while the soil moisture content increased or decreased compared with the control at each periods of flue-cured tobacco growth in 2016 and 2017,and no significant difference was found. The soil bulk density increased by 0.083 percentage points and the porosity decreased by 3.113 percentage points at 30 days after transplanting in 2015 after continuous application of biochar,but no significant difference was found,the soil bulk density decreased by 0.010-0.144 percentage points and the porosity increased by 0.500-5.537 percentage points at each periods in other years after continuous application of biochar,and the effects of biochar on the soil bulk density and porosity were significant difference at 30 days after transplanting in 2017. Continuous application of biochar had little impact on the ratio of solid phase of soil at each periods in 2015 and 2016,which could significantly reduced the ratio of solid phase of soil at 30 days after transplanting in 2017. Continuous application of biochar could extremely significantly increased the ratio of liquid phase of soil at each periods in 2015,and reduced the ratio of gas phase of soil at 30 days after transplanting in 2015,while had little impact on the ratio of solid phase and ratio of gas phase of soil at each periods in other years. The results of principal component analysis of the index of soil physical properties in each period of different years showed that the soil physical properties were obviously different from the control after continuous application of biochar,which was beneficial to the improvement of soil physical properties.

In order to study the influence of different application amounts of charcoal on the microbial diversity in tobacco rhizosphere soil, with the rhizosphere soil of flue-cured tobacco variety Yunyan 87 as the research object, we compared the situation by applying different amounts of charcoal, and conducted high-throughput sequencing of rhizosphere soil ITS2 region and 16S rDNA-V4 region with progressive treatment with 3 application amounts of charcoal; through bioinformatics analysis of data, we obtained the OTU abundance, distribution,α diversity, community species composition and abundance information of microorganisms in rhizosphere soil when different treatment methods were used, and conducted PCA clustering analysis and UPGMA clustering analysis of the community composition and abundance. The results showed that within the application scope of 50-150 g/tree, by increasing the application amounts of charcoal, it could increase the variety diversity and distribution uniformity of bacteria in rhizosphere soil, while it would reduce the variety diversity and distribution uniformity of fungus; compared to the control group where no charcoal was applied, after applying the charcoal with the amounts of 50, 100,150 g/tree, the abundances of Proteobacteria bacteria had declined by 4.1%, 2.7% and 0.7% respectively; the abundances of Acidobacteria bacteria increased by 10.4%, 8.1% and 7.7% respectively; the abundances of Actinobacteria bacteria were all lower than the abundance of control group; the difference in abundance of Gemmatimonadetes bacteria between various groups was very small. After applying the charcoal with the amount of 50 g/tree, the abundance of Zygomycota fungus had declined by 12.38% compared to the control group(50.92%), and then, with the increased of the application amount of charcoal, the abundance of Zygomycota fungus gradually increased to 53.68%; on the contrary, after applying the charcoal with the amount of 50 g/tree, the abundance of Ascomycota fungus had increased by 10.15% compared to the control group(30.63%), and then, with the increased of the application amount of charcoal, the abundance of Ascomycota fungus gradually declined to 29.11%; after applying the charcoal with the amount of 50 g/tree, the abundances of Basidiomycota and Chytridiomycota funguses almost had no change, and then, with the increased of the application amount of charcoal, the abundances of Basidiomycota and Chytridiomycota funguses presented the trend of increased first, and then declined; various treatment methods did not have significant influence on the abundance of Glomeromycota fungus. By studying the influencing method and action rules of charcoal application on the micro-ecology of tobacco rhizosphere, it can provide theoretical basis to the application of charcoal in tobacco field.

In order to study the feasibility of using biomass fuel in tobacco curing,on the base of early research results,the text used the formula 50% wood chips plus 50% tobacco stem as materials to produce 8 mm-diameter pellet fuel by an efficiency biomass pellet machine(HCK045A).Five treatments were used during tobacco curing,including T0(100% coal) as contrast,T1(30% biomass fuel plus 70% coal),T2(50% biomass fuel plus 50% coal),T3(70% biomass fuel plus 30% coal) and T4(100% biomass fuel).Indexes including dry and wet bulb temperature,comprehensive thermal efficiency,chemical components of flue-cured tobacco leaves,pollutants content of combustion gas,economic characters and curing cost of different treatments were analyzed.The results showed that,the curing barn which used biomass fuel warned up more quickly during yellowing stage and leaf-drying stage than the curing barn which used coal but slower during stem-drying stage and the thermal efficiency was higher.The chemical components of cured tobacco leaves were more suitable and the coordination was better.The content of main pollutant SO₂ of combustion gas from the curing barn which used biomass fuel reduced significantly,especially the content of treatment T4 only was 4.17% of CK.The curing cost increased because the increasing of labor and materials,and the range of T1 was lowest(1.72%) but that of T4 was highest(20.62%).However,due to the higher ratio of mid-high grade leaves the output values of curing treatments with biomass fuel all increased,especially the range of T1 was highest and extended 22.91%.

In order to study the effect of drought stress on physiological characteristics of grafting flue-cured tobacco,using KRK26 as scion, KRK26,K326 and Anyan 2 as rootstock,analysing and discussing root morphology, plant morphology and physiological indexes of resistance in pot culture under different treatment. The results indicated that under drought stress conditions at early period, the relative content of chlorophyll (SPAD readings) decreased, while the content of proline, superoxide dismutase (SOD) activities, catalase (CAT) activities and peroxidase (POD) activities in flue-cured tobacco demonstrated an increasing trend. The root length,surface area,plant height,stem girth, node spacing and LAI of the grafted tobacco plants showed a decreasing trend,the content of proline continued to increase in both grafted seedlings and non-grafted seedlings with the aggravation of drought stress when tobacco leaves were turning to mature, whereas, the SPAD readings, SOD activities, CAT activities and POD activities all demonstrated a decreasing trend. The speed of decreasing in different root grafted was slower than that in own root grafted. Compared with KRK26/K326 and KRK26/KRK26, KRK26/Anyan 2 had greater parameters under drought stress, but the SPAD readings did not have significant difference. All the results showed that less damage was made to KRK26/Anyan 2 than KRK26/K326 and KRK26/KRK26 under drought stress, and KRK26/KRK26 suffered the most serious damage. According to correlation analysis, there was a certain correlation between morphological indexes and resistance indexes of grafting flue-cured tobacco. In conclusion, there were obvious differences among grafted tobacco in physiological characteristics, and there response to soil drought. The order of drought resistance was KRK26/Anyan 2, KRK26/K326 and KRK26/KRK26 in turn from strong to weak, based on the main root morphology,plant morphology,physiological characteristics under drought stress.

2C protein phosphatase (PP2C) is a key component in the ABA signal transduction pathway and plays a pivotal role in plant growth and development,cell cycle regulation and adaptation to adversity stresses.To explore the function of the PP2C gene in tobacco adaptation to abiotic stress,a PP2C homologous gene was cloned from the tobacco cultivar K326,which contained a 1 617 bp ORF encoding 538 amino acid. The homology analysis showed that the gene had high homology with PP2C16 of Nicotiana tomentosiformis. So it was named NtPP2C16. Bioinformatics analysis showed that the NtPP2C16 catalytic region had 11 structural sub-regions which were relatively conservative in the PP2C family evolution. qRT-PCR analysis revealed that the expression of this gene was significantly induced by ABA and H₂O₂ signal molecules,and responded to drought,high salt,low temperature and low potassium stresses. NtPP2C16-pBI121 overexpression vector was constructed successfully,the results provided some basis for analysis of NtPP2C16 responsing to abiotic stresses in tobacco.