Isolation, Identification And Biodegradation Characteristics Of Precocene Ⅱ Degrading Bacteria

Ageratum conyzoides, an annual herbaceous plant, originates from central America, which has occurred in south and south-west of China and becom a regional nixious weed. It could produce kinds of allelochemicals and often become dominant species in invaded ecosystem. A. conyzoides mainly release allelochemicals to environment through volatilization and more than 80% of volatile substances are PrecoceneⅠand PrecoceneⅡ(Precocene 130.93%, PrecoceneⅡ51.61%).What is more, the allelopathic activity of PrecoceneⅡis stronger than PrecoceneⅠ. Therefore, we chose PrecoceneⅡas object. For the restoration of ecosystem invaded by exotic allelopathic plants, the key problem is how to weaken even eliminate their allelopathic effects on native plants. Former researches suggested that microorganisms using allelochemicals as sole source of carbon and energy existed in the soil under allelopathic plants, which could degrade or transform allelochemicals in the process of metabolism.Our study based on the above theory, the object of which was to isolate allelochemical degrading microorganisms from soil beneath A. conyzoides.We chose PrecoceneⅡas representative of allelochemicals of A. conyzoides. Above all, a method for determination of precoceneⅡwas developed by HPLC. Then PrecoceneⅡ-degrading strains were isolated from the soil invaded by A. conyzoides in Hainnan Province. The optimal degrading conditions were obtained through studying seven factors(incubation time, incubation concentration, temperature, rotation rate, initial PH, additional carbon source and nitrogen source) affecting strains growth and degrdation ratio to PrecoceneⅡ, meanwhile, the effects of combination between key influence factor on strains'biodegradation were investigated in the paper. The results were summerized as follows:First, The PrecoceneⅡsamples were analyzed on a Luna C18 column (150mm×4.6 mm,5 um) using methyl alcohol-ultrapure water (80:20, V/V) as the mobile phase at a flow rate of 1 mL·min-1. The detection wavelength was 224 nm and the column temperature was 35℃. The injection amount was 10μL every time.Second,21 Precocene II degradable strains were isolated from A. conyzoides-invaded soil by using mineral salt media(MSM) added Precocene II as solo carbon source. The degradation ratio of Precocene II by the strains was tested with HPLC. Seven of the strains could degrade Precocene II to a certain extent and one (bacterial strain X2)of them showed potential for further ultilization with a degradation ratio of PrecoceneⅡby 51.21% incubating 26h.Third, basing on analysis of phenotype, physiological and biochemical characters and 16srDNA, the bacterial strain X2 was gram-negative.Through identification, strain X2 was most similar to Novosphingobium capsulatum(Sphingomonas capsulate)and the homology index was up to 99.1%.Fourth, the effects of incubation time, incubation concentration, temperature, rotation rate, initial PH, additional carbon source and nitrogen source on degradation were determined. The results showed that:The degrading rate of strain X2 slowed down in 26h. The optimal temperature of strain X2 degrading PrecoceneⅡwas 25℃and the optimal PH was 7.5. The degradation capacity of X2 increased with adding additional carbon source and when lactose was added the biodegradation rate was higher than other additional carbon source. Using different nitrogen sources, the higher biodegradation rate appeared when yeast powder and peptone were used. However, incubation concentration and rotation rate had no effects on the biodegrdation.Furthermore, the effects of combination between key factor on strains'biodegradation were studied. From single factor experiment, we knew that temperature, initial PH, additional carbon source and nitrogen suorce were key factors. The results indicated that:Obvious correlation was showed by the effects of any two-key-factor-combination on biodegradation. Under the combination of temperature and initial PH, the highest biodegradation rate-60% appeared at 25℃,PH7 or PH 7.5. Under the combination of temperature and additional carbon source, the highest biodegradation rate-70% appeared at 25℃or 30℃with any additional carbon source. Under the combination of temperature and nitrogen source, the highest biodegradation rate-67% appeared at 25℃with yeast powder or peptone as nitrogen source. Under the combination of PH and additional carbon source, the highest biodegradation rate-75% appeared at PH7 or PH7.5 with any additional carbon source. Under the combination of PH and nitrogen source, the highest biodegradation rate-72% appeared at PH7 or PH7.5 with yeast powder or peptone as nitrogen source. Under the combination of additional carbon source and nitrogen source, the highest biodegradation rate-79% appeared at lactose as additional carbon source and yeast powder or peptone as nitrogen source.