摘要
本研究在纯培养条件下,对9种外生菌根真菌(Ectomycorrhizal fungi, ECM真菌)灰鹅膏菌Amanita vaginata、乳黄粘盖牛肝菌Suillus lactifluus、绒粘盖牛肝菌Suillus tomentosus、亚黄粘盖牛肝菌Suillus subaureus、灰环粘盖牛肝菌Suillus laricinus、美丽褶孔牛肝菌Phylloporus bellus、褐黄牛肝菌Boletus luridus、黄地勺菌Spathularia flavida和粘盖牛肝菌Suillus bovinus在柴油胁迫下的耐受性进行了筛选;并从石油烃污染地采样分离可降解柴油的菌根真菌促生细菌,对菌根真菌-菌根促生细菌互作关系作了研究,得出以下结论:
1外生菌根真菌柴油降解能力的筛选
对9种外生菌根真菌的柴油降解能力进行了筛选,在9种ECM真菌中柴油对Suillus tomentosus(ST)的抑菌率均显著低于其余各菌;无论是在液体含油培养基还是葡萄糖含油培养基中,ST均表现出很高的菌丝生物量和柴油降解率;采用GC/MS对ST柴油降解后的产物分析可得,碳数为18-29的正链烷烃被降解的较多,降解产物多为碳数较少的正链烷烃、含多个侧链及侧链较长的烷烃。在含油培养基中添加少量葡萄糖可促进菌丝生物量在柴油胁迫下的积累,显著降低柴油对各菌的抑菌率,提高柴油降解率。
2可降解柴油的菌根促生细菌的筛选
在石油烃污染地的油松菌根上分离出可在以柴油为唯一碳源的无机盐培养基上生长的细菌18株。将18株细菌与菌根真菌ST在纯培养条件下对峙培养,筛选出对菌根真菌生长具有促进作用的细菌2株及抑制作用较低的细菌3株;通过ST与上述5株细菌在Kottke液体培养基中的共培养,筛选出菌株S826及H2对ST生物量的积累有显著的促进作用。
3纯培养条件下单、双接种对柴油降解效率的影响
在10 g/L柴油含量的液体培养基中研究了单接种ST、S826、H2及ST-S826与ST-H2双接种的柴油降解率,其降解率分别为36.6%、22.7%、41.4%、63.9%及55.3%,双接种ST-H2柴油降解效率显著高于单接种ST及单接种H2;双接种ST-S826柴油降解效率显著高于单接种ST与单接种S826之和。S826可显著促进菌根真菌ST对柴油的降解效率。
4促生细菌S826及H2的菌种鉴定
S826在KB培养基上为优势菌株,菌落边缘光滑,呈黄色,有凸起,粘稠而有光泽,表面湿润,KB培养基变成绿色,有恶臭。革兰氏反应阴性,椭圆状。硫化氢试验、甲基红试验、硝酸还原试验、吐温水解试验和明胶液化试验为阴性;吲哚试验,运动性试验、柠檬酸盐试验为阳性。16S rDNA PCR产物长度均为1.5 kb,系统进化树显示,菌株S826与GU186116.1(Pseudomonas putida strain IHBB1369)菌株构成一个分支,16S rRNA序列的同源性达到99%以上。基于生物学特性和16S rDNA序列分析,将S826初步定为恶臭假单胞菌属中的已知种。
H2在锰营养琼脂培养基上为优势菌株,菌落呈浅黄色,微微隆起,有光泽,易挑起,边缘齿状。革兰氏反应阳性,菌体杆状,有芽孢。硫化氢试验、吐温水解试验、柠檬酸盐试验为阴性;吲哚试验,运动性试验、甲基红试验、硝酸还原试验和明胶液化试验为阳性。系统进化树显示,菌株H2与EU931557.1( Brevibacillus brevis strain ZFJ-2)菌株构成一个分支,16S rRNA序列的同源性达到99%以上。基于生物学特性和16S rDNA序列分析,将H2初步定为短短芽孢杆菌属中的已知种。
5菌根真菌及其促生细菌提高油松降解柴油的作用
实验设油松单接种ST(S)、S826(B)及双接种ST与S826(S+B)3个处理,研究不同处理的油松幼苗在0、2、5、10 g/kg干土柴油浓度下的生理反应。结果表明接种处理的油松幼苗(B、S与S+B)生长状况(株高、地径、油松幼苗生物量、根冠比)优于未接种对照;单、双接种处理通过提高油松体内酶活性(SOD)、脯氨酸、叶绿素、可溶性蛋白含量、根系活力及降低植物体内MDA含量减缓了柴油胁迫对植物的毒害作用,有效提高了柴油的降解效率。在柴油初始浓度为2 g/kg干土时,接种处理B、S与S+B的柴油降解效率分别为79.8%、81.5%与82.9%,分别为对照(74.5%)的107.1%、109.4%和111.3%;在柴油浓度为5 g/kg干土时,接种处理的柴油降解效率分别为71.7%、77.1%与81.1%,显著高于对照(69.9%);柴油浓度为10 g/kg干土时,接种处理柴油降解效率为61.7%、66.1%与71.2%,分别比对照(54.2%)高13.8%、22.0%、31.4%。同一柴油浓度下,双接种处理柴油降解效率显著高于单接种,单接种处理S柴油降解效率显著高于单接种处理B。表明双接种处理柴油降解效率优于单接种,单接种菌根真菌处理S优于单接种细菌处理B。
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 diesel
9 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 diesel
18 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 culture
Single 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 H2
S826 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. brevis
5 The effect of ST associated with its MHB on improvement pinus’s ability to degrade diesel
This 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.
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