Researches On The Mechanisms Of Different Cultivation Measures On Relieving Tobacco Continuous Monoculture Problems

With Nicotina tobaccum L.cv.'K326' used as experimental materials,this study adopteddifferent planting patterns (multiple crop rotation and multiple continuous monoculture mode) anddifferent soil management methods (kept fallow in winter, rice straw planted in winter, Astragalussinicus planted and then returned to field in winter) to discuss their effects on relieving continuousmonoculture problems in the tobacco production. To explore the underlying mechanism, the yieldand quality of tobacco, the structure and function diversity of rhizospheric microorganisms, soilenzyme activities, soil toxicity and root differential proteomics were carried out in this study,which provided the theoretical basis and the technical support for recovering and improvingproductivity of the tobacco-planted soil. The main research results were as following:1. Mechanisms of different planting patterns on relieving tobacco continuous monocultureproblemsThe results showed that multiple crop rotation mode (MR) was favorable for the growth oftobacco, and could increase the tobacco yield and quality and the proportion of high qualitytobacco. Besides, the MR soil has the weaker autotoxic effect. Using agar plate counting, thenumber of rhizospheric soil bacteria, fungi and actinomycetes under MR are higher than that ofmultiple continuous monoculture mode (MC). BIOLOG analysis indicated that Average WellColor Development (AWCD) of carbohydrates, amino acids, fatty acid, amino acids, phenolic acid,and carboxylic acid of MR were all higher than that of MC. Furthermore, the diversity indices ofmicrobes utilization of carbon sources suggested that the microbial functional diversity, such asSHANNON, EVENNESS, BRILLOUIN indices, in rhizospheric soil of flue-cured tobacco wererobust under MR treatment. Among the soil enzyme activities, protease, urease, dehydrogenase,cellulase, sucrase and acidic phosphatase activities of MR were higher than that of MC, and theirdifferences reached significant level. However, peroxidase and catalase activities of MC werehigher than that of MR.The results showed that a total of21protein spots in tobacco root at late vigorous growingstage had significant expression difference;14protein spots were identified using LC MS/MSanalysis and database searching. Among them, three proteins were related to aroma formation,i.e. transketolase, monodehydroascorbate reductase(MDHA), O-methyltransferase-like protein. All were up-regulated under MR, so the tobacco quality was better under MR. Eight proteins wereinvolved in energy metabolism, plant antioxidation reaction, translocation of solute, metabolism ofnucleic acid. They are phosphoglycerate kinase, mitochondrial import inner membrane translocasesubunit Tim13, NBS-LRR type protein, insecticidal crystal protein(ICP), DnaJ protein, Vesicletrafficking protein Sly1, Ribosomal protein-like protein, Mg-dependent phosphatase. Front sevenprotein under MR were highly expressed and thus promoted the growth of tobacco. However, theywere down-regulated under MC.In conclusion, MR treatment was favorable to ameliorate the stability of the soilmicro-ecological system and improve rhizospheric soil nutrient equilibrium, promote tobacco rootgrowth, and boost the growth of tobacco, increase the yield and quality of tobacco, and gain thegood economic benefit. To a certain extent, MR treatment lighten continuous monocultureproblems.2. Mechanism of different soil management methods on relieving tobacco continuousmonoculture problemsThe results showed that the soil treated with rice straw that was returned to field (RS) has theweakest autotoxic effect. BIOLOG analysis indicated that the RS treatment was beneficial for thegrowth of the microflora feeding on amines as the carbon sources. Furthermore, principalcomponent analysis (PCA) from Biolog data showed that the components1and2(PC1, PC2)which were extracted from31factors related to the utilization of carbon sources could explained52.9%and47.1%of variance respectively, and carbon sources of carboxylic acids, phenolic acidsand amines play the main contribution role in the the separation of principal components. Thediversity indices of microbes utilization of carbon sources suggested that the microbial diversity,such as SIMPSON, SHANNON, SHANNON evenness, BRILLOUIN, McIntosh indices, inrhizospheric soil of flue-cured tobacco were robust under RS treatment. The activities of soilenzymes related to the circulation of soil nutrition, such as urease, protease, cellulase and sucraseetc., were higher under RS and AS treatments than that under WF treatment. The activities of soiltexture improving related enzymes, such as polyphenol oxidase, peroxidase, catalase,dehydrogenase etc., were higher under WF or AS treatments than that under RS treatment.The results showed that a total39protein spots in tobacco root at late vigorous growing stage displayed significantly different expression as compared to the control;24protein spots wereidentified using LC MS/MS analysis and database searching. Among them, two proteins wererelated to aroma formation, i.e. transketolase, GDP-mannose3',5'-epimerase(GME). They wereup-regulated under RS treatmen, so it boosted the tobacco quality. But they were down-reguatedunder AS treatment, so the tobacco quality was worse under it. Kinase-associated proteinphosphatase(KAPP), putative transposon protein, leucine-rich repeat protein kinase-likeprotein(LRR-RLK), Aspartyl protease family protein, Mitogen-activated protein kinasekinase(MAPK), Rab3GTPase-activating protein catalytic subunit, Vesicle trafficking protein Sly1,E3ubiquitin-protein ligase BAH, alpha-hydrolase domain-containing protein, acetyl-CoAsynthetase(ACS), ATPase splayed, β-glycosidase-like and so on, they were involved in signaltransduction, translocation of solute, protein metabolism, energy metabolism. They were veryimportant for growth development of tobacco and all were up-regulated under RS and AStreatments. They were all down-regulated under WF and thus tobacco grew weakly. In conclusion,RS treatment was favorable to improve rhizospheric soil nutrient equilibrium of fule-curedtobacco and ameliorate the stability of the soil micro-ecological system, promote tobacco rootgrowth, boost the growth of tobacco, increase the yield and quality of tobacco, and gain the goodeconomic benefit. And RS treatment lighten continuous monoculture problems to some content.The results revealed that different cultivation measures with the purpose of improvingbiodiversity, including the planting pattern of multiple crop rotation mode (tobacco-rice→rice-rice→tobacco) and soil management method that rice straw planted in winter and then returned tofield (RS), were conducive to recover and reconstruct well rhizosphere ecological system oftobacco, and relieve tobacco continuous monoculture problems.