Detection of Paenibacillus Polymyxa by Fluorescent in Situ Hybridization and Its Application in Pig Slurry Compost and Soil
|School||Nanjing Agricultural College|
|Keywords||Paenibacillus polymyxa fluorescent in situ hybridization (FISH) spore organic fertiliter soil|
P. polymyxa belongs to the group of plant growth promoting rhizobacteria (PGPR). The effects of P. polymyxa have been reported including e.g., nitrogen fixation, soil phosphorus solubilization, production of antibiotics, auxin, chitinase, and hydrolytic enzymes, as well as promotion of increased soil porosity. Many reasearchers have been reported P. polymyxa is non-pathogenic to animal and human, and some strains are disease-resistant. P. polymyxa SQR21 was applied to produce bio-organic fertilizer, and its had good effect to control plant diseases. In order to understand the behavior of P. polymyxa in manure and soil, fluorescent in situ hybridization (FISH) was used to identify its vegetable cell and spores, and the procedure of FISH was optimized. This research will provide scientific basis to the detection of P. polymyxa in bio-organic fertilizer and environmental condition. The results obtained are listed as follows:1. The optimal detection conditions of P. polymyxa SQR21 by FISH are listed as follows:Samples washed by 1×PBS twice to three times when collecting, fixed by ethanol at 4℃for 2 h, dispersed by 0.01% SDS. The hybridization temperature and time were 48℃and 2 h, respectively. The concentrations of formamide in hybridization buffer and sodium chloride in washing buffer were 35% and 60 mM, respectively.2. Two methods of chemical substance pretreatment and germinant pretreatment were selected to detecting P. polymyxa SQR21 spores. Chemical substance pretreatment methods including 10 M HCl at 37℃for 30 min,0.1 M NaOH at 0℃for 30 min, and 10 mg/mL SDS+50 mM DTT at 60℃for 15 min,7 mg/mL lysozyme at 37℃for 15 min, the hybridization efficiencies were 75.2%,86.6% and 64.7%, respectively. Germinant pretreatment by L-alanine + adenosine + casamino acids at 37℃for 5 min, and the hybridization efficiency was 92.3%. The results of scanning electron microscopy showed that the structure of spore was loose and lysed at some degree treated by chemical substance, while the structure of spore was smoth and intact treated by germinant pretreatment.3. The result of detecting P. polymyxa SQR21 inoculated in pig slurry compost by germinant pretreatment FISH and plate counting method simultanously showed that the numbers of total cell and spore of FISH were ten times higher than those of plate counting method. FISH could reflect the change of spores more exactively.4. The result of detecting P. polymyxa SQR21 in compost stored at room temperature by FISH showed that the number of P. polymyxa significantly increased after fermented two days and three days. The spores were visible after five days. The number of total cell reduced, but the number of spores increased relatively and most of the cells were spore after ten days. The number of total cell was changeless and the spores were absolute predominant after twenty days.