| Polycyclic aromatic hydrocarbons (PAHs) are a class of environmental organic pollutants that are considered potentially extremely harmful owing to their toxic, mutagenic, and carcinogenic characteristics. A better understanding of how plants take up and metabolize PAHs will benefit the risk evaluation of plant PAH contamination. Recently, utilizing endophytic bacteria to modulate the metabolism of organic pollutants in plant and reduce the risk of plant organic pollution has attracted much attention. The use of endophytic bacteria to regulate the metabolism of organic pollutants and to reduce contamination risks in plants would be significantly advantageous. Assessing the diversity, distribution, physiology, and ecology of endophytic bacteria in plants is prerequisite for isolating organic contaminant-degrading endophytic bacteria and using them to eliminate organic pollution in plants. However, to our knowledge, little information is available about endophytic bacterial populations and PAH-degrading genes in plants from PAH-contaminated sites.In this paper, the distributions of endophytic bacteria and some PAH-degrading genes in Alopecurus aequalis Sobol and Oxalis corniculata L. grown in soils contaminated with different levels of PAHs were investigated. By using organic solvent extraction combined with high performance liquid chromatography (HPLC) determination, polymerase chain reaction followed by denaturing gradient gel electrophoresis technology (PCR-DGGE), and traditional cultivation methods, we get the following conclusions:(1) The cultivable endophytic bacteria detected in the two tested plants were about 104 to 107 CFU per gram (fresh weight), and the number of endophytic bacteria in roots was higher than that in shoots. A total of 68 endophytic bacterial strains were isolated from different tissues of the two plants and classified into three phyla:Firmicutes, Proteobacteria and Bacteroidetes. Interestingly, although the cultivable endophytic bacterial populations of two plants shows low diversity, Bacillus spp. and Pseudomonas spp. were the shared dominant cultivable populations. With the PAH pollution level increased, the number of cultivable endophytic bacteria decreased rapidly, meanwhile, the diversity and distribution of endophytic bacteria in the two plants changed correspondingly. Most isolated endophytic bacteria could grow well on Luria-Bertani media in the presence of different PAHs, and some isolates can even grow rapidly on the mineral salt medium with a single PAH as the sole carbon or energy source, indicating that these strains may have the potential to degrade these PAHs.(2) The PCR-DGGE results showed that the endophytic bacterial communities in the roots and shoots of the two plants were quite different; pollution intensity, host plants and plant tissues could significantly influence the diversity of the endophytic bacterial community. With the pollution level strengthened, the Shannon-Wiener indexes of two plants were decreased, indicating that the plants grown in lightly polluted soils showed the higher diversity.77 different bacterial 16S rRNA gene V3 sequences were obtained from the DGGE gel, sequenced and blasted. The phylogenetic analysis indicated that these bacteria belong to four phyla (Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes), and Proteobacteria was the predominant flora.(3) The naphthalene dioxygenase (NAH) gene and phenol monooxygenase (PHE) gene were two degrading genes involved in the catabolic metabolism of PAHs in plants. The PCR-DGGE indicated that most plant tissues in PAH-contaminated sites carried NAH and PHE genes, and the diversity of PHE gene was higher than that of NAH gene.48 bands representative for the PAH-degrading genes were got from the DGGE gel, among which 9 were identified as NAH gene, and the others were PHE gene. |
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