Effect Of Ectomycorrhizal Fungi Associated With Mycorrhizal Helper Bacteria On Improvement Pinus Tabulaeformis Ability To Degrade Diesel

This paper was conducted for investigating the capability of ectomycorrhizal (ECM) fungi Amanita vaginata,Suillus lactifluus,Suillus tomentosus,Suillus subaureus,Suillus laricinus,Phylloporus bellus,Boletus luridus,Spathularia flavida and Suillus bovinus to degradate diesel in vitro. The mycorrhizal helper bacteria (MHB) able to degradate the diesel oil were isolated and purified from ectomycorrhizal of Pinus tabulaeformis, Study on the interaction between ectomycorrhizal and MHB was carried out. Conclusions as follows:1. Screening of the ECM fungi to degrade diesel9 species of ECM fungi were exposed to culture contained diesel, the inhibition of diesel on Suillus tomentosus ( ST) was significantly lower than the rest. ST have shown a high mycelial biomass and diesel oil degradation efficiency in Kottke medium with diesel oil as sole carbon source whether or not added glucose; analysis of production of diesel after 14 days biodegradation by ST using GC / MS, most 18-19 Cs normal paraffin hydrocarbons were degraded to paraffin hydrocarbons with much less Cs,multi-branched and long-branched hydrocarbonSThe mycelial biomass and the rate of diesel degradation were effecicenty promoted when some glucose (1 g/L) was added in the diesel-only Kottke medium.2. Isolating and screening MHB able to degrade diesel18 strains of bacteria were isolated from the ectomycorrhizal of Pinus tabulaeformis grown in petroleum contamined soils using inorganic salt medium with diesel as sole carbon source. Under face-to-face culture with ST, Screening out 2 strains which can promote ST growth and 3 have less inhibition. These five strains co-culture with ST in Kottke liquid medium, only strains S826 and H2 can promote ST biomass accumulation apparently.3. Effect on diesel degradation efficiency of single and double inoculation under pure cultureSingle inoculation with ST, S826, H2, and double inoculation with ST-S826 and ST-H2 in Kottke medium with diesel oil as sole carbon source (10 g/L) , the efficiency of diesel degradation were 36.6%, 22.7%, 41.4%, 63.9% and 55.3%. The degradation efficiency of double inoculation ST-H2 was much higher than single inoculation ST and H2, and ST-S826 was higher than the sum of single inoculation ST and S826 significantly. S826 can promote the diesel degradation efficiency of ST apparently.4 Identification of Strains S826 and H2S826 was the dominant strain in KB medium, colony is yellow, viscous and shiny, with central uplift, surface moist and edges smooth, KB medium was turned into green and fetid. Gram-reaction: negative. Hydrogen sulfide, methyl red test, nitrate reduction test, Tween hydrolysis and gelatin liquefaction test were negative; Indole test, exercise test and the citrate test was positive. The phylogenetic tree showed that strains S826 and GU186116.1 (Pseudomonas putida strain IHBB1369) formed a branch, 16S rRNA sequence homogeneity among this two strains was more than 99%. Based on biological characteristics and 16S rDNA sequence analysis, S826 was identified as P. putida.H2 is the dominant strain in manganese nutrient agar medium, colony is pale and yellow, bright, with central slightly elevated, easy to provoke, edge dentate. Gram-positive, spore-forming and rod-shaped bacteria. H2 Hydrogen sulfide test, Tween hydrolysis test and citrate test were negative; indole test, exercise test, methyl red test, nitrate reduction test and gelatin liquefaction teste were positive. Phylogenetic tree showed that strains H2 and EU931557.1 (Brevibacillus brevis strain ZFJ-2) constitute the same branch, 16S rRNA sequence homogeneity among this two strains was more than 99%. Based on biological characteristics and 16S rDNA sequence analysis, H2 was identified as B. brevis5 The effect of ST associated with its MHB on improvement pinus's ability to degrade dieselThis experiment was set pinus single inoculated with bacterial S826 (B),ST (S) and dual inoculated with ST-S826 (S+B), the control group only pinus Studying on pinus's Growth and physiological responses under these four treatmenrs in soils containing 0,2,5,10 g diesel per kilogram dry soil. The pot trial result in the treatments B,S and S+B's pinus growth status (height, ground diameter, biomass and root to shoot ratio of pinus seedlings) superior to treatment CK The single and dual inoculation treatments could improve enzyme activity (SOD), proline, chlorophyll and soluble protein content, root activity and lower MDA content in plants to inhibit diesel toxcity on pinus and promote the efficicency of diesel degradation. The diesel degradation efficicency of B,S and S+B was 79.8%, 81.5% and 82.9% separately when 2 g diesel in per kilogram dry soil, is 107.1%,109.4% and111.3% of CK (74.5%);69.9%, 71.7%, 77.1% and 81.1% while 5 g diesel in per kilogram dry soil and 10 g diesel in per kilogram dry soil was 61.7%, 66.1% and 71.2% separately, and 13.8%,22.0%,31.4% higher than CK (54.2%). At the same diesel concentration, the degradation efficiency on diesel of S+B was higher than S and B, and S is higher than B.