Construction Of Compound Microorganism Against Hytophthora Parasitica Var. Nicotianae

Phytophthora parasitica var. nicotianae is one of the most important pathogen causing hugedamage to yield and quality of tobacco in recent years. We isolated soil bacteria from Jimo county ofQingdao. First, the antagonistic bacteria were selected by antagonizing action and anti-disease test.Second, we separated and purificated the bacteria for nitrogen fixation, phosphorus solubalization andpotassium solubalization from soil by the selective medium, and mixed fermentation, got obtainingstable bacterial mixture having good promoting effect. Last, it was found that stable bacterial mixturewith the most obvious antibiological effect. The results as following:1. A total of92strains of bacteria were isolated from Tobacco rhizosphere soil. And26of themshowed antagonistic effects against Phytophthora parasitica var. nicotianae. Results of furtherscreening showed that7strains exhibited strong antagonistic action. At last, Integrated the diseasescontrol for the pot tobacco,4bacteria were selected for further studied, numbered22,24,3-4, and5-2.They were identification based on the results of morphological observation and16SrDNA sequence test.The result showed that strain22was Bacillus subtilis. Strain24was Bacillus vallismortis. Strain3-4was Alcaligenes faecalis. Strain5-2was Bacillus amyloliquefaciens.2. We separated and purificated the bacteria for nitrogen fixation, phosphorus solubalization andpotassium solubalization from soil by the selective medium.28bacterial strains with ability of nitrogenfixation,phosphorus solubalization or potassium solubalization were mixed and inoeulated to12medias,respectively,and incubated to get mix cultures,which were inoculated to the same media andthen incubated under the same condition and repeated for about10times. In each culture whichobtaining stable bacterial mixture contains two or three species. In pots,10stable bacterial mixturesobserved significant growth promotion effects after being inoculated to upland tobacco. Using agarplate method,7mixed cultures of the stable bacterial mixture exhibited signifieant growth inhibition toplant pathogens such as Phytophthora nicotianae. It was found that22+6A,22+7B and5-2+11A werethree stable bacterial mixture with the most obvious antibiological effect.3. A Pot experiment was carried out to quantitatively investigate the dynamic change ofPhytophthora parasitica var. nicotianae in tobacco rhizosphere by the method of Real-time PCR. Thereal-time PCR results showed that the copy numbers of gene in tobacco rhizosphere with no treated wasmuch higher than that in the treatments with compound microorganism. The results indicated that thecompound microorganism can reduce the number of the pathogen in tobacco rhizosphere, thuseffectively controlled the tobacco black shank.4. Compound microbial composition were identification based on the results of morphologicalobservation and16SrDNA sequence test. The result showed that strain6A1was Enterobacter cloacae.Strain6A2was Enterobacter aerogenes. Strain7B1was Acinetobacter tandoii. Strain7B2wasWautersiella falsenii. Strain7B3was Enterobacter asburiae.