Inoculation Of A Phenanthrene-degrading Endophytic Bacterium Reduces Phenanthrene Contamination And Alters The Bacterial Community Structure In Wheat

Polycyclic aromatic hydrocarbons(PAHs)are a group of persistent organic pollutants that are found extensively in contaminated soil in our country.PAHs in soil can be taken up and accumulated by plants,and then enter the food chain,which represents a serious threat to crop safety and human health.Therefore,how to reduce the PAHs contamination risk in plant have been widely concerned.Recently,endophytic bacteria for regulating and controlling the uptake and metabolism of PAHs in plants have been a focus of interest.They can colonised and proliferated in the interior of plant,promote plant biomass and possess the capacity to degrade organic contaminants in plant.However,there is little information available to indicate how PAH-degrading endophytic bacteria influence the endophytic microbial communities and degradation enzyme activity of plant grown in PAH-contaminated soils.Thus,the results of this work will improve the PAHs biodegradation and metabolism of the plant and reduce the residual of plant PAHs contamination.In this study,an efficient phenanthrene-degrading endophytic bacterium Massilia sp.Pn2,was used for colonization into wheat tissues(Triticum aestivum L.cv.Yangmai-16).Initially,the antibiotic-resistance of stain Pn2 and plant growth-promoting properties were explored.Then,the influences of strain Pn2 colonization on wheat growth and phenanthrene biodegradation were performed using greenhouse experiments.More importantly,the effect of strain Pn2 inoculation on the endophytic microbial communities and degradation enzyme activity of plant grown in PAH-contaminated soils were also systematically investigated.The main results were shown as follows:1.Inoculation of strain Pn2 reduces the phenanthrene concentration and accumulation in wheat.Inoculation of strain Pn2 reduced the phenanthrene concentration,accumulation and the translocation factor in wheat.Strain Pn2 was resistant to ampicillin(20 mg·L-1)and chloramphenicol(20 mg·L-1).So the two antibiotics were selected to mark strain Pn2 after colonization on root surface of wheat.Results shown that strain Pn2 can colonised and proliferated in the interior of wheat,promote the wheat growth,and increased the wheat biomass with producing indoleacetic acid(IAA)and siderophore.The presence of large numbers of Pn2 in wheat tissues reduced the level of phenanthrene concentration.For example,after 30 days,the respective Pn2 counts in wheat roots and shoots were 3.74 to 4.53 log cfu·g-1 FW and 2.88 to 3.38 log cfu·g-1 FW;Compared with the Pn2-free treatment,the respective concentrations of phenanthrene in endophyte-colonized roots and shoots were 15.5-28.4%and 20.8-30.6%lower,respectively.Meanwhile,inoculation of strain Pn2 can also reduce the phenanthrene contaminzation in soil.2.Colonization of endophytic bacterium strain Pn2 influences the endophytic bacteria community in wheat.The total DNA of the entire endophytic bacterial community in wheat roots and shoots was extracted,and the Illumina PE250 sequencing technology was conducted to explore the endophytic bacterial diversity and community structure in wheat.Results shown that phenanthrene contamination and strain Pn2 colonisation determined the endophytic bacterial community structure in wheat.Although the endophytic bacterial community structure was various different in wheat roots and shoots,all of the bacterial phyla and genera in wheat shoots were also found in wheat roots,and the majority phylum of endophytic bacteria in wheat were Proteobacteria,and the dominant genus was Pseudomonas sp..Low and moderate level of phenanthrene contamination increased the richness and diversity of endophytic bacteria in internal wheat tissues.Furthermore,inoculation of strain Pn2 increased the richness and diversity of endophytic bacteria in wheat roots and shoots.For example,the richness of endophytic bacteria in wheat significantly increased in the presence of low level of phenanthrene contamination(p<0.05).Meanwhile,inoculation of strain Pn2 also influenced bacterial phyla and genera in wheat roots and shoots.For example,Pn2 inoculation increased the relative abundance of Firmicutes compared with that in the absence of Pn2 inoculation irrespective of the contamination level;and the relative abundance of Pseudomonas sp.in wheat roots was higher than that in those not inoculated with Pn2 in S0-S2 soils,however,the relative abundance of Pseudomonas sp.in wheat shoots was markedly lower than that in wheat shoots not inoculated with Pn2 in different treated soils(p<0.05).3.Colonization of endophytic bacterium strain Pn2 alters the enzyme activities in wheat.Inoculation of strain Pn2 influenced the activity of polyphenol oxidase(PPO)and peroxidase(POD)in wheat.Results shown that that phenanthrene contamination and strain Pn2 colonisation determined the enzyme activities in wheat.The activity of PPO and POD increased first and then decreased in wheat with Pn2-free as the phenanthrene contamination increasing.When the phenanthrene concentration was 50 mg·kg-1,the activity of PPO and POD in wheat with Pn2-free were the highest.Inoculation of strain Pn2 increased the activity of PPO and POD in wheat.For example,the activity of PPO in wheat roots significantly increased(p<0.05)with inoculation of Pn2 after 20 days;the activity of POD in wheat roots significantly increased(p<0.05)with inoculation of Pn2 after 20 and 40 days.