| ABSTRACT:Tobacco is one of the important economic crops in our country and also the main economic sources of the national and local governments. Tobacco bacterial leaf spot diseases, such as tobacco wildfire and tobacco angular leaf spot,which often occur in the tobacco production have brought enormous economic loss sometimes.But these diseases are difficult to prevent beacause of the influence of cultivation, climate, drugs, etc and the common chemical control can easily cause drug resistance and environment pollution. In order to explore new control way for these diseases, it is quite necessary to study dynamics of infection of pathogenic bacteria in leaf surface and interaction between bacterial leaves diseases occurrence and Leaf surface microecology.The research method based on PCR such as semi-quantitative PCR and real-time quantitative PCR(RTQ-PCR) can accurately, sensitively and quickly detect the dynamic changes and quantification of pathogenic bacteria during infections in plant.PCR-DGGE technique can analysis leaf surface bacteria communities and their dynamics changes. It may be a disease prevention strategy of regulate leaf surface bacterias by screening material for regulating bacteria quorum sensing or biofilm formation.In this study, mutant strain of Pseudomonas syringae pv. Angulata(Kanr) was inoculated into maturity tobacco leaves in pot culture. Selective medium and specific PCR were analyzed dynamic changes and quantification of pathogenic bacteria during infections in leaf surface. PCR-DGGE technique was analyzed leaf surface bacteria communities and their dynamics changes. Some salt compounds, Amino acids, organic acidic compounds, ellagic acids and carbohydrate compounds were analyzed influence of pathogenic bacteria and leaf surface bacterias biofilm formation.The main results of this study were followed:1. In this study, EZ-Tn5transposon-mediated P. s.pv. Angulata(Kanr) mutant H048was shake culture for12～14h in KB culture medium, then collected bacterias, and prepared bacterial suspension by aseptic water (OD600=0.5).Then bacterial suspension were inoculated malture period tobacco leaves by spraying method, water was control, setted4repetitions, sampled leaf disk at different times. Selective medium KB (concentration of Kan is50μg/ml) and common medium PDA were counted respectively of pathogenic bacteria and leaf surface bacterias. The results showed that dynamic changes of pathogenic bacteria is first increased then decreased. Bacterial accumulation was observed at3d, then quantification is increased to maximum at5d and then decreased.Extracted DNA and diluted101～107to reserve. Semi-quantitative PCR was detected dynamics of infection ofpathogenic bacteria in DNA. Highly conservative16S rDNA gene was the reference gene, amplified16SrDNA gene by PCR with primers Eubac10F/1507R. The specific primers of P. s. pv. Angulata HrpZ359/1115were amplified pathogenic bacteria by PCR. Then quantity one tested optical density of electrophoresis strip. The results showed that results of Semi-quantitative PCR is the same as bacterial plate count. Dynamic changes of pathogenic bacteria is first increased then decreased and the quantification of pathogenic bacteria is maximum at5th.2. In this study, bacterial plate count was used to detect interaction between bacterial leaves diseases occurrence and Leaf surface microecology. The results showed that the changes of total leaf surface bacteria was the same to dynamics of infection of pathogenic bacteria. The dynamic change is first increased then decreased. The changes of total leaf surface bacteria is stable.In this study, PCR-DGGE technique was used to analysis leaf surface bacteria communities. The results showed that the bands of DGGE and distribution are different between inoculated leaves and water control. The bands of DGGE to water control is more than inoculated leaves.3. In this study, pathogenic bacteria H048and isolates such as111013-4,111013-6,111013-8,120106-12was screen suitable growth conditions to bacteria biofilm by air-liquid of KB,1/2KB,1/10KB, LB, MMFM culture medium. The results showed that is suitable for biofilm formation in KB culture medium. The biofilm of H048,111013-4,111013-6,111013-8and120106-12was observed in KB culture medium at7d.The results also show that111013-4,111013-6,111013-7can formate biofilm quickly than H048in the same condition. There is obvious biofilm at3d. H048and111013-7were used to screen17compunds effcted biofilm formation. Include9kinds of salt compounds,5kinds of amino acids,1kind of organic acidic compound,1kind of ellagic acid and1kind of carbohydrate compound.The results showed that different compunds have different effction for bifilm formation of H048and111013-7. There are14kinds of compunds are influential for bifilm formation of H048and111013-7in experimented17kinds of compunds. Among them, succinic acid, ellagic acid, Co2+, EDTA2-, D-Try, D-Leu, D-Tyr and D-Met can inhibit bifilm formation of H048and111013-7. however the concentration of succinic acid is2.00mg/mL, it can inhibit bifilm formation of H048but the inhibition to biofilm formation of111013-7is become weak.NH4+, Mg2+and Glucose only promote bifilm formation of H048and the concentration is1.25×101mg/ml.Mn2+, Cu2+and B4O72-can inhibit bifilm formation of H048and111013-7, or promote bifilm formation of H048. When the concentration of Mn2+is1.25×101mg/ml, it can inhibit bifilm formation of111013-7and promote bifilm formation of H048. When When the concentrations of Mn+are decreased to1.25mg/ml and1.25×10-1mg/ml, they only can promote bifilm formation of H048. When the concentration of Cu2+are1.25×101mg/ml and1.25mg/ml, they can inhibit bifilm formation of H048and111013-7.When When the concentrations of Cu2+is decreased to1.25×10-1mg/ml, it only can promote bifilm formation of H048. When the concentration of B4O72-is1.25×101mg/ml, it can inhibit bifilm formation of H048and111013-7.When the concentrations of B4O72-is decreased to1.25mg/ml, it only can promote bifilm formation of H048.Bacteria biofilm is a important structure for infected in host plants and the different bacteria can formate differently biofilm. In this study, the results proof that111013-4,111013-6,111013-7can formate biofilm quickly than H048in the same condition. In the17kinds of biofilm formation regulation compounds, when the concentration of succinic acid is2.00mg/mL, it can inhibit bifilm formation of H048but the inhibition to biofilm formation of111013-7is become weak. It is a kind of important compound to be used in pot culture and field experiment. We want to control diseases by regulate biofilm formation and Leaf surface microecology.In this study, interaction between bacterial leaves diseases occurrence and leaf surface microecology is researched and Some compounds were analyzed influence of pathogenic bacteria and leaf surface bacterias biofilm formation at preliminary. It will be a new control way for tobacco bacteria leaves sopt by regulate biofilm formation and Leaf surface microecology. |
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