Microorganism Mechanism Of Continuous Cropping Obstacles Of Rehmanmia Glutinosa And Development Of Beneficial Microbial Inoculant

Rehmannia glutifnosa is a perennial herb,and its root is considered to be of high medical value in traditional Chinese medicine.Unfortunately,this plant suffers from very serious continuous cropping obstacles and can not be continuously cultured in the same field till 8-10 years of other plants cropped which greatly limits the yield and quality of R.glutifnosa.In this study,we explored the changes in microbial community structure of rhizosphere soil of R..glutifnosa first and continuous cropped when disease occurred.using high-throughput sequencing and quantitative PCR.Then,we isolated some pathogenic microorganism to R.glutifnosa,and investigate the antagonistic effect of several plant probiotics on these pathogens.In previous research,we have developed a microbial inoculant which can effectively promote roots swelling of R.glutifnosa in lab-scale condition.Based on above results,we improved our microbial inoculant with above bacteria that could inhibit the pathogenic microorganism.And we performed two years field experiment to test the effect of the original and improved inoculant on overcoming continuous cropping problem of R.glutifnosa.The main results are shown as the following aspects:(1)The results of high-throughput sequencing of microbes in the rhizosphere soil of healthy and diseased R.glutifnosa first and continuous cropped showed that there were significant differences in microbial communities in rhizosphere soil between healthy and diseased R.glutifnosa both first and continuous cropped.Proteobacteria,Acidobacteria and Firmicutes were the dominant bacteria.Ascomycota was the dominant fungi.At the level of genus,Sphingomonas was dominant in all samples.The contents of beneficial bacteria such as Streptomyces in the rhizosphere soil of diseased R.glutifnosa were lower than that in the rhizosphere soil of healthy R.glutifnosa from the first and the second year plant,while Pseudomonas indicated the opposite trend.The fungal community of different samples was quite different.In the rhizosphere soil of first year planted healthy R.glutifnosa,Monographella and Thielaviopsis almost accounted for 50% of the total fungi,while Un-s-Hypocreales sp,Un-s-Nectriaceae sp etc.had advantage in the rhizosphere soil of the first year planted diseased R.glutifnosa.In the second year plant,Un-sectinaceae sp,Un-s-Sordariales sp etc.were abundant in rhizosphere soil of healthy R.glutifnosa,while Un-s-sordariales sp was dominant and its content was almost 41% in diseased R.glutifnosa rhizosphere soil.According to results of the first year and the second year plant,the content of Un--s-Ascomycota sp in the rhizosphere soil of diseased R.glutifnosa was higher than that in the rhizosphere soil of healthy R.glutifnosa,while Monographella indicated the opposite trend.The abundance of total bacteria,total fungi,Pseudomonas,Sphingomona and F.oxysporum in rhizosphere soil of healthy and diseased R.glutifnosa were quantified by quantitative real-time PCR.The results showed that the ratio of total bacteria to total fungi in rhizosphere soil decreased from 7.63×109 to 8.70×106 with the disease of R.glutifnosa,suggesting that the soil transformed from “bacterial type” to “fungal type”.The abundance of Pseudomonas and F.oxysporum in rhizosphere soil of diseased R.glutifnosa was significantly higher than that in the rhizosphere soil of healthy R.glutifnosa,while the abundance of Sphingomona in rhizosphere soil of diseased R.glutifnosa was lower than that in rhizosphere soil of healthy R.glutifnosa.(2)We isolated pathogens from the diseased root of R.glutifnosa and obtained three strains of mold,two strains of bacteria and one strain of yeast.The reincoluate experiment showed that all six microbes were pathogenic,espacailly Rhizopus stolonifer,Rhodotorula paludigena and Pseudomonas hibiscicola caused 100% diseased rates.Then we used plant probiotics to confront the microbes with high morbidity.The result showed that the probiotics had weak inhibitory effect on Rhizopus stolonifer and Enterobacter aerogenes while strong inhibitory effect on Septoria digitalis Pass,Fusarium solani and Rhodotorula paludigena.(3)In the first year field experiment,R.glutifnosa were treated with differerent microbial inoculants.The average root weight per plant was determinate.The weight of the treatment 1,2 and 3 was 188.194 g per plant,84.3495 g per plant,82.3175 g per plant,respectely,which were significantly higher than the control of 56.3475 g per plant.Although the weight of treatment 4 was lower than the control,there were not abvious differences between the two.These results showed that the application of our microbial inoculant in a suitable way can effectively increase the yield of R.glutifnosa in continuous cropping conditions.The results of the second year field experiment indicated that the new microbial inoculant siginificantly increased the average root weight of “jingjiu”,a kind of R.glutifnosa.