Development of a stable attenuated double-mutant of tobacco mosaic virus for cross-protection

Tobacco(Nicotiana tabacum)and tomato(Solanum lycopersicum)are two major economic crops in China.Tobacco mosaic virus(TMV;genus Tobamovirus)is the most prevalent virus infecting both crops.Currently,some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus.Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases.However,stable attenuated TMV mutants are scarce.In this study,we found that the substitutions in the replicase p126,arginine at position 196(R^(196))with aspartic acid(D),glutamic acid at position 614(E^(614))with glycine(G),serine at position 643(S^(643))with phenylalanine(F),or D at position 730(D^(730))with S,significantly reduced the virulence and replication of TMV.However,only the mutation of S^(643) to F reduced the RNA silencing suppression activity of TMV p126.A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants.Furthermore,our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants.This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.

Variations of phyllosphere microorganisms in asymptomatic and tobacco brown spot leaves before and after spraying 12% difenoconazole + fluxapyroxad SC

12%difenoconazole+fluxapyroxad SC(commercial name:Jiangong)was first released by BASF in China in 2016.It has been registered to control many diseases,including pear scab,apple Alternaria leaf spot,tomato early blight,cucumber powdery mildew,etc.This study evaluated the bioactivity of Jiangong against Alternaria alternata and explored variations of phyllosphere microorganisms in both asymptomatic and tobacco brown spot leaves at different persistence periods(0,5,10,and 15 days post-fungicide application)using high-throughput sequencing technology.The results indicated that Jiangong effectively inhibited mycelial growth(average EC_(50) value of 0.51μg/mL),conidia germination(average EC_(50) value of 3.47μg/mL),and the carbon metabolism of A.alternata.Both asymptomatic and symptomatic leaves presented complex microbial communities.Higher fungal diversity was noted in asymptomatic leaves,while higher bacterial diversity was found in symptomatic leaves.After application,the diversity and abundance of microbial community structures in both types of leaves changed over time.Fungal microbiome communities showed greater sensitivity than bacterial groups,with the microbiome communities of asymptomatic leaves being more affected than those of symptomatic leaves.Fungal community diversity decreased for both symptomatic and asymptomatic leaves after 5 days of application,while the diversity of fungal community in symptomatic leaves showed an upward trend after 10 days of application.Meanwhile,bacterial community diversity increased in both symptomatic and asymptomatic leaves after 5 days of application but then declined in asymptomatic leaves after 15 days.The abundance of the dominant function group of phyllosphere bacteria(metabolism,genetic information processing,environmental information processing)was not affected by the application of Jiangong.However,the abundance of the dominant function group of phyllosphere fungi(animal pathogen-endophyte-wood saprotroph,endophyte-plant pathogen,plant pathogen-undefined saprotroph)was significantly affected by the application of Jiangong,and high variation was found in symptomatic leaves than that of asymptomatic leaves.The application of Jiangong-induced alterations in the community structure of the tobacco phyllosphere microbiome provides a basis for future tobacco brown spot control strategies based on phyllospheric microecology.

Study on Microsatellite Distribution in Complete Genomes of Tobacco Vein Clearing Virus

MATLAB software and optimal complete subgraph algorithm were used to extract and reveal the microsatellite distribution features in the complete genomes of the tobacco vein clearing virus （NC-003 378.1） from the NCBI database.The results showed that the repetitions number and their location of the N-base group has been extracted and displayed.The largest repetitions of N-base group in the complete genomes of the tobacco vein clearing virus was decreased as the exponential function with the increasing of N.The method used in this study could be applied to the extraction and revealing of the microsatellite distribution features in the complete genomes of other viruses,thereby provided a basis for the research of the structure and the law of function,inheritance and variation by the using of the microsatellite distribution features.

Variation of Enzyme Activity in Flue-cured Tobacco-growing Soil Planted with Different Lastseason Crops

The rice soil （last crop was rice） and arid red soil （last crop was corn） were used as a test material for the pot experiment. The variation of enzyme activi-ty in flue-cured tobacco-growing soil planted with different last-season crops was in-vestigated at different growth stages in this study. The results showed the activity variation of the 3 enzymes differed in the 2 soils at different growth stages. The catalase activity in the arid red soil trended to decrease overal from the vigorous growing stage to harvesting stage; while it decreased gradual y in the rice soil until the harvesting stage. The phosphatase activity in the 2 soils al increased with the proceeding of growth period. The urease activity in the arid red soil decreased gradual y at different growth stages, but the variation of urease activity in rice soil was irregular. During the growth of flue-cured tobacco, the catalase and urease ac-tivity in the arid red soil increased first and then decreased, and the phosphatase activity increased gradual y. ln rice soil, the catalase activity increased first and then decreased; the phosphatase activity decreased first and then increased; the urease activity increased first, then decreased and increased last. The activity of al the en-zymes in the 2 soils showed significant differences compared to the control except some enzymes at the vigorous growing stage. lt was suggested the planting of flue-cured tobacco would affect greatly the soil enzyme activities.

Investigation and Analysis of Continuous Cropping of Flue-cured Tobacco in Henan Province

The continuous cropping of flue-cured tobacco in the major producing areas of Henan Province by designing questionnaire, fieldwork, enquiry and phone calls, mails, and other forms, the data were obtained then classified and analyzed. The results showed that the main planting areas of flue-cured tobacco were in Nanyang, Xuchang, Luoyang, Sanmenxia, Pingdingshan, and Luohe where were the major producing areas of succession cropping of flue-cured tobacco with percentage of 20%-40% for three years and over three years, and a few places up to 50%. Morbidity was common with wide distribution, and the main diseases were black shank, viral disease, and black rot. It proved that tobacco was not suitable for continuous cropping. Both of tobacco disease and pest rate and mortality significantly increased by more than 40% under continuous cropping for three years and over three years, and the yield and quality of flue-cured tobacco also decreased after continuous cropping. For many places, the tobacco yield started to decrease in the second year of continuous cropping, and after three-year cropping, the yield decreased in the range of 9%-24%. Continuous cropping for a long time would make a serious decline in the quality of tobacco, resulting in non-economic benefits. Therefore, in order to reduce the occurrence of diseases and economic loss in tobacco production, it is recommended to avoid continuous cropping, or to adopt appropriate measures to control tobacco continuous cropping obstacles in the unavoidable case.

Study on Effect of Engran Series of Microbial Organic Fertilizers on Flue-cured Tobacco

[Objective] This study was conducted to provide theoretic support for improving yield and quality of flue-cured tobacco by applying microbial organic fertilizers. [Method] A field plot experiment was conducted to study the effects of the Engran series of microbial organic fertilizers on growth and development, diseases and pests and economic traits of flue-cured tobacco. [Result] The result showed that the treatment of conventional fertilization ＋ Engran microbial agent could promote the growth and dry matter accumulation of flue-cured tobacco during the growth period in the field, enhance the disease resistance of tobacco, and improve yield and output value of flue-cured tobacco and income of tobacco growers. Compared with T1,the proportion of high-grade tobacco, the proportion of mid-high grade tobacco,yield, output value and average price increased by 4.4%, 3.8%, 5.48%, 11.51% and5.73% respectively. [Conclusion] The treatment of conventional fertilization ＋ Engran microbial agent showed outstanding performance, is optimum for field production,and could be applied to Flue-cured tobacco.

Effects of Microbial Fertilizers in Improving Acidic Tobacco-planting Soil and Tobacco Leaf Quality

[Objectives]This study was conducted to improve acidic soil and enhance the quality of tobacco leaves.[Methods]The effects of different microbial fertilizers on improving acidic tobacco-planting soil and tobacco leaf quality were investigated through plot experiments.[Results]The application of microbial fertilizers could improve the pH value of acidic soil,and composite microbial agent A showed the best application effect.The application of bio-organic fertilizer was beneficial to improving the contents of available phosphorus and available boron in acidic soil.The application of composite microbial agent A was beneficial to improving the contents of available phosphorus and available potassium in acidic soil,and could promote the growth of tobacco plants and improve the economic traits of flue-cured tobacco and the coordination of chemical components in tobacco leaves.The application of composite microbial agent B led to a downward trend in the content of available boron in acidic soil.The application of composite microbial agent B could promote the absorption of nutrients by tobacco plants,and improve their disease resistance and the quality of tobacco leaves.Due to the differences in functional microorganisms contained,the application effects of different microbial fertilizers in improving acidic tobacco-planting soil and improving tobacco leaf quality varied.Overall,the application of microbial fertilizers could increase soil pH,activate soil nutrients,promote tobacco growth,enhance disease resistance,increase tobacco output value,and improve tobacco quality.Microbial fertilizers have good application prospects in improving acidic soil and improving tobacco quality.[Conclusions]The application of microbial fertilizers to improve acidic tobacco-planting soil can ensure the normal growth and development of tobacco plants and the improvement of tobacco leaf quality,achieving high-quality and sustainable development of Zhaotong tobacco.

Effects of Green Manure Mixed Cropping Patterns on Physical and Chemical Properties of Soil and Economic Characters of Flue-cured Tobacco

[Objective] The aim was to clear the suitable green manure cropping pat- terns in Xiangxi tobacco-planting areas. [Method] 8 treatments were set to study the effects of the monoculture and mixed cropping of common vetch （Vicia gigantea Bge.）, perennial ryegrass （Lofium） and rapeseed （Brassica campestris L.） on physi- cal and chemical properties of soil and economic characters of flue-cured tobacco. [Result] （1） Green manure turnover can reduce soil bulk density by 1.08%-8.62%, and the effect of green manure mixed cropping pattern was the best. （2） Green manure turnover also can increase the soil nutrient, soil organic matter, total nitro- gen （N）, total phosphorus （P）, total potassium （K）, alkali-hydrolyzale N, rapidly available P and rapidly available K by 1.44%-6.10%, 0.01-0.12 g/kg, 1.89%- 11.32%, 0.12%-3.56%, 1.06%-11.76%, 0.04%-18.93% and 0.98%-23.12%, respec- tively, and the effect of the monoculture of common vetch was the best.（3） The overall change of soil pH was not obvious.（4）Green manure turnover can increase the yield and output of flue-cured tobacco, and the effect of the monoculture of common vetch was the best. [Conclusion] The monoculture of common vetch can be generalized in the dry land of Xiangxi tobacco-planting areas.

Effects of intercropping vines with tobacco and root extracts of tobacco on grape phylloxera, Daktulosphaira vitifoliae Fitch

The effects of grape-tobacco intercropping patterns on populations of grape phylloxera, Daktulosphaira vitifoliae Fitch, as well as on the growth and development of the infested vines were evaluated in the field and the impact of an aqueous tobacco root extract on grape phylloxera was evaluated using a laboratory bioassay. The aqueous tobacco root extract exhibited biological activity against this pest. The egg mortality, nymph mortality, development period, life span and female fecundity were significantly affected. In the field trial, grape phylloxera populations were clearly lower as compared to the monoculture pattern. However, the rates of newly developed roots and newly infested grape roots were significantly higher and lower, in intercropping patterns than in the vine monoculture, respectively. The grape phylloxera population number on the grape roots decreased each year, and the vine trees gradually renewed upon continuous intercropping with tobacco over three years. These results confirmed that intercropping grapes with tobacco can effectively control grape phylloxera in an infested vineyard. The results also indicated that additional crops could be intercropped with grapes and are effective against grape phylloxera, which should be explored as an integrated approach for controlling the pest.

SIMYB1 and SIMYB2, two new MYB genes from tomato, transcriptionally regulate cellulose biosynthesis in tobacco

Cellulose, a major constituent of plant biomass, is synthesized by a cellulose synthase complex. It has been demonstrated that MYB genes transcriptionally regulate cellulose synthase in Arabidopsis. However, little is known about this process in tomato. Here, two MYB （SIMYB1/2） and three cellulose synthase （CESA） （SICESA41516） genes were isolated. SIMYB1/2 and SICESA4/5/6 accumulation was found to correspond to cellulose accumulation in different tissues of tomato. Dual luciferase assays indicated that these two MYBs were transcriptional activators that interact with promoters of SICESA4/5/6. Moreover, SIMYB2 could also activate promoters of SIMYB1/2, suggesting the possible underlying auto-activation mech- anisms for MYB transcription factors. Transient over-expression of SlMYB1/2 in Nicotiana tabacum up-regulated tobacco endogenous NtCESA genes and increased cellulose accumulation. The function of SIMYB112 was further investigated using stable transformation and the results indicated that N. tabacum lines heterologous expressing SIMYB1/2 displayed a pleiotropic phenotype, long and narrow leaves, with NtCESA induced and significant increase of cellulose. In conclusion, our data suggest that tomato SIMYB1/2 have transcriptional regulatory roles in cellulose biosynthesis and SIMYB2 was more effective than SIMYB1, which may due to the transcriptional activation by SIMYB2 on SIMYB1 and itself.

Control Effect of Different Microbial Agents on Tobacco Bacterial Wilt

[ Objectives] The paper was to study effects of different microbial agents on occurrence of tobacco bacterial wilt. [ Methods I Antagonistic strains against Ralstonia solanacearum were screened from in situ soil, and prepared into four different combinations. The number of soil microorganisms and incidence of bacterial wilt of four combinations were observed. [ Results] The incidence rate of bacterial wilt treated by microbial agent OR-1 was significantly lower than that in control, and the control efficacy against bacterial wilt reached 67%. [ Conclusions] Application of microbial agent OR-1 could effectively reduce the incidence rate and disease index of bacterial wilt, thus improving the quality of tobacco.

Comparative transcriptome analysis reveals differential gene expression in resistant and susceptible tobacco cultivars in response to infection by cucumber mosaic virus

Cucumber mosaic virus(CMV) is one of the most severe viral diseases transmitted by aphids infecting Solanum crops in China, causing great losses of crop yields and income in rural communities.The tobacco cultivars NC82 and Taiyan 8 are closely related but differ in resistance to CMV.NC82 is susceptible to infection and Taiyan 8 is resistant, but the mechanisms underlying this difference in resistance are not clear.In this study, we conducted RNA sequencing to analyze changes in gene expression induced in the leaves of Taiyan 8 and NC82 upon systemic infection with CMV, compared with gene expression in the leaves of mock-inoculated plants.Leaves were sampled at one, three, eight, and 15 days after infection.In total, 3443 and 747 differentially expressed genes were identified in Taiyan 8 and NC82, respectively.Gene ontology and pathway enrichment analyses revealed that the different responses to CMV infection between cultivars were based on microtubulebased processes, pentose and glucuronate interconversions, plant–pathogen interaction,and hormone signal transduction pathways.Genes encoding pathogenesis-related proteins, disease-resistance proteins, lipoxygenase, cellulose synthase, an auxin response factor, and an ethylene receptor showed different expression patterns.The differences in gene expression following CMV infection likely contributed to the different resistance levels of these two tobacco cultivars.The comprehensive transcriptome dataset described here,which includes candidate response genes, will serve as a resource for further studies of the molecular mechanisms associated with tobacco defense responses against CMV.

Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

Effects of Bio-organic Fertilizer with Antagonistic Bacteria against Tobacco Bacterial Wilt on Soil Microbial Communities and Disease Resistance of Tobacco

In this study, the effects of bio-organic fertilizer with antagonistic bacteria against tobacco bacterial wilt on soil microbial communities and disease resist- ance of tobacco were investigated by field experiment. The results showed that the incidence of tobacco bacterial wilt in bio-organic fertilizer treatments （T3 and T4） decreased remarkably among four treatments in the field. Compared with the local conventional fertilization group, the incidence of tobacco bacterial wilt was re- duced by 21.9% and 25.0% in T3 and T4, respectively ; the yield of flue-cured tobacco was improved by 5.7% and 5.3%, respectively ; the proportion of mid- high grade tobacco leaves increased by 2.3% and 2.6%, respectively. After application of bio-organie fertilizer with antagonistic bacteria against tobacco bacterial wilt, rhizosphere soil microbial communities exhibited vast amount and abundant species ; the amount of rhizosphere soil bacteria of infected tobacco plants was im- proved by 218.5% with fewer species. It could be concluded that the application of bio-organic fertilizer with antagonistic bacteria against tobacco bacterial wilt could improve the ecological environment of tobacco field, inhibit the growth of pathogenic bacteria, decrease the incidence of tobacco bacterial wilt, and enhance the quality of flue-cured tobacco. This study laid the foundation for further ecological prevention and control of soil-borne diseases of tobacco.

Integration of GC-MS Based Non-Targeted Metabolic Profiling with Headspace Solid Phase Microextraction Enhances the Understanding of Volatile Differentiation in Tobacco Leaves from North Carolina, India and Brazil

In this report, gas chromatography-mass spectrometry (GC-MS) based non-targeted metabolomics is used to develop appropriate headspace solid phase microextractions (HS-SPME) to enhance the understanding of volatile complexity of flue-cured tobacco leaves. Non-targeted metabolic profiling of GC-MS shows that the extraction condition of HS-SPME at 100?C for 30 min provides a better metabolite profile than other extraction conditions tested. GC-MS and principal component analyses (PCA) show that among five types of fibers tested, 100 μm polydimethylsiloxane (PMDS), 65 μm polydimethylsiloxane/divinylbenzene (PMDS/DVB) and 75 μm carboxen/polydimethylsiloxane (CAR/ PMS) provide a better reproducible metabolite profile. Based on an appropriate PDMS extraction condition optimized, we use GC-MS analysis and PCA to compare metabolite profiles in flue-cured leaves of tobacco plants grown in North Carolina, India and Brazil, respectively. The resulting data of PCA show that the global metabolic profiles in North Carolina samples are separated from those in Brazil and India samples, two groups of which are characterized by a partially overlapped pattern. Several peaks that were differentially accumulated in samples were annotated to known metabolites by deconvolution analysis, such as norsolanadione, solavetivone and rishtin. Norsolanadione is detected only in Brazil samples. Solavetivone is detected in samples of India and Brazil but not in those of North Carolina. Rishtin is detected in samples of North Carolina and India but not in Brazil samples. These data indicate that not only can a non-targeted metabolic profiling approach enhance the understanding of volatile complexity, but also can identify marker volatile metabolites in tobacco leaves produced in different growth regions.

Comparison of simultaneous distillation extraction and solid-phase micro-extraction for determination of volatile constituents in tobacco flavor

The volatile and semi-volatile components in tobacco flavor additives were extracted by both simultaneous distillation extraction and solid-phase micro-extraction. Extraction conditions for solid-phase micro-extraction were optimized with information theory. Then, detection were accomplished by gas chromatography-mass spectrometry. Characteristic of each method was compared. Qualitative analysis and quantitative analysis of 6# tobacco flavor sample were accomplished through both simultaneous distillation extraction and solid-phase micro-extraction. The experimental results show that solid-phase micro-extraction method is the first choice for qualitative analysis and simultaneous distillation extraction is another good selection for quantitative analysis. By means of simultaneous distillation extraction, 20 components are identified, accounting for 92.77% of the total peak areas. Through solid-phase micro-extraction, there are 17 components identified accounting for 91.49% of the total peak areas. The main aromatic components in 6# tobacco flavor sample are propanoic acid, 2-hydroxy-, ethyl ester, menthol and menthyl acetate. The presented method has been successfully used for quality control of tobacco flavor.

Use of Real- time RT-PCR Analysis for mRNA Expression of Tobacco Ferritin Gene (NtFer1)

To understand the use of real-time reverse transcription-polymerase chain reaction （real-time RT-PCR） for detecting the relative abundance of mRNA, the expression of a tobacco ferrltin gene （NtFer1） was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 cleady and similady, namely NtFer1 expression was responsive to iron-ovedoad, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.

Judgment of Aroma Types of Middle and Upper Flue-cured Tobacco Leaves Based on Stepwise Discriminant Analysis

[Objective] The aim of this study was to establish mathematical models for judging the aroma types of middle and upper flue-cured tobacco leaves according to the contents and proportions of aroma compositions. [Method] The aroma types of tobacco leaves were judged based on stepwise discriminant analysis, using 63 C3F and 65 B2F tobacco leaf samples from 13 tobacco producing regions in 11 provinces of China （Huili in Sichuan, Baokang in Hubei, Wulong in Chongqing, Lu- oyang in Henan, Zhucheng in Shandong, Wuyi Mountain in Fujian, Malong in Yun- nan, Chuxiong in Yunnan, Bijie in Guizhou, Liuyang in Hunan, Suiyang in Guizhou, Kaiyuan in Liaoning, Nanxiong in Guangdong） as calibration samples, and 67 aroma components as indices. And the Fisher discriminant functions were verified using 21 C3F and 19 B2F tobacco leaf samples. [Result] Variation coefficients of the propor- tions were lower than that of contents of most aroma components in middle and upper leaves of the samples, indicating that the proportions were more stable than contents of aroma components. The proportions of benzyl alcohol, solanone, β-dam- ascone, neophytadiene, farnesylacetone A, palmitic acid, thunbergol, methyl linole- nate and cembratriene-diol were all over 1% in both middle and upper leaves, al- though the dominant aroma components of the same aroma type varied between middle and upper leaves. Moreover, 11, 18, 7 and 11 aroma components were re- spectively introduced into the Fisher discriminant functions established based on the contents and proportions of middle and upper flue-cured tobacco leaves, which ex- hibited accuracy rates of 91.7%, 100%, 91.7% and 91.7% in the judgments of other tobacco leaf samples. The results revealed that the components those determined aroma types in middle leaves were obviously more than in upper leaves. In middle leaves, the accuracy rates of aroma type judgment could be improved by using the proportions rather than the contents of aroma components as indices. However, the functions based on the proportions and the contents of aroma components in upper leaves gave close accuracy rates. [Conclusion] The results of the study will provide references for identifying aroma types of flue-cured tobacco leaves in future work.

Integrated Biological Control of Tobacco Bacterial Wilt (<i>Ralstonia solanacearum</i>) and Its Effect on Rhizosphere Microbial Community

Biological control agents and soil amendments have been applied to control tobacco bacterial wilt, but the mechanism is not well-known. In the present study, a field experiment was performed to investigate the soil physicochemical properties, disease index (DI) and disease incidence of tobacco bacterial wilt, and rhizosphere microbial community. The results show that the control efficacy of single application of antagonistic bacteria and calcium cyanamide was 46.43% and 51.92%, respectively. While the combined control efficacy of antagonistic bacteria and calcium cyanamide was 65.79%. Besides, the combined application of antagonistic bacteria and calcium cyanamide could increase soil pH, total N alkaline N, and exchangeable Ca, which were negatively associated with the microbial diversity, soil-borne pathogenic microorganisms, and incidence of tobacco bacterial wilt. Additionally, the combination of antagonistic bacteria and calcium cyanamide can improve the proportion of some antagonistic microbial species, and these antagonistic microbial species were inversely associated with the DI of tobacco bacterial wilt. In conclusion: The integrated measure may influence soil microbial communities through enhancing soil physicochemical properties and rebuild healthy soil microbial community structure to mitigate tobacco bacterial wilt. The current study presented valuable insights into the mechanisms enhancing soil health in the integrated measure.

Changes in Soil Enzyme Activity and Nutrient Content in Different Years of Continuous Cropping Tobacco Fields

In order to find out changes in the main fertility indicators of continuous cropping tobacco fields in Henan Province with years of continuous cropping,5 typical tobacco fields( Jia County,Shaoling District,Fangcheng County,Xiangcheng County,and Song County) were studied. Indicators in question included activity of main enzymes( sucrase,urease,catalase,and acid phosphatase),p H value,organic matters,total nitrogen,available phosphorus and available potassium. The results indicate that in most tobacco fields,the activity of sucrase and urease increased in two years of continuous cropping,later,it showed a declining trend with increase in years of continuous cropping; in some fields,it directly declined with increase in years of continuous cropping; the activity of catalase increased in two years of continuous cropping,later,it showed a declining trend with increase in years of continuous cropping,and it became stable after three years of continuous cropping;generally,the activity of acid phosphatase declined with increase in the years of continuous cropping within 3 years continuous cropping,later,it became stable. Except Fangcheng County,the soil p H value of other districts( counties) generally declined with increase in years of continuous cropping,and it was in the range of 5. 6-6. 8. With the increase in continuous cropping,the soil organic matters( SOM) in tobacco fields of Jia County and Song County declined,changes of SOM in tobacco fields of Fangcheng County and Xiangcheng County were not obvious,and SOM showed the trend of first decline then rise in Shaoling District. In most tobacco fields( Jia County,Xiangcheng County,and Song County),the total nitrogen showed a declining trend; in Shaoling District,it showed a trend of first decline then rise; in Fangcheng County,it showed a rising trend. The available phosphorus firstly slightly declined then rose in tobacco fields of Shaoling District and Song County; it showed a trend of first rise then decline in Jia County; there were no obvious changes in Fangcheng County and Xiangcheng County. Changes in the available potassium were different in tobacco fields. In sum,there are certain rules for changes in soil enzyme activity and nutrient content in tobacco fields in Henan Province,but changes are not completely the same. It is concluded that the site conditions and farming activities exert a certain influence on soil enzyme activity,p H value,organic matters,and nitrogen,phosphorus and potassium content. Therefore,to solve obstacles in continuous cropping of tobacco fields,different regions should take different measures.