| Objective To explore the mechanism of the continuous cropping obstacles in the process of Chrysanthemum morifolium planting and the molecular mechanism of alleviating continuous cropping obstacle,we analyzed the production of Chrysanthemum morifolium,physical and chemical properties of rhizosphere soil,rhizosphere bacteria and endophytic bacteria of Chrysanthemum morifolium organs.Methods 1 Using wild field trials,six treatments were arrayed—Chrysanthemum morifolium was cultured in rotation cropping field(rotation cropping group),except this group other five were set on the field which was already continuously mono-cropped for tow consecutive years with Chrysanthemum morifolium—continuous mono-cropping group,continuous mono-cropping with application of fermented silkworm excrement group,with application of silkworm excrement group,with application of bacterial manure group,with application of lime group.2?Using Miseq for analyzing the bacterial community structure differences between fermented silkworm and silkworm.3?In harvest period,recording survival rate,plant height,branch number and yield of each group and rhizosphere soil samples was collected for the physical and chemical properties analyzing 4?Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism(T-RFLP).5 Rhizosphere bacteria were examined using T-RFLP and Miseq.Results 1?The result showed that microbial diversity increased and the bacteriastructure obviously changes with the fermentation of silkworm excrement.The content of Proteobacteria increased from 22.05%to 53.09%and promoted the growth of Bacteroidetes andVerrucomicrobia,weakened the growth of Firmicutes and Actinobacteria.Through the analysis of the genus,we found that the beneficial bacteria of silkworm excrement were biocontrol bacteria(Baccillus and Burkholderia),while in fermented silkworm excrement except these there were azotobacter(Devosia?Nitratireducto and Rhizobium and Ochrobactrum which can degrade Allelochemicals.2?The survival rate of continuous mono-cropping group was 80%,other 5 groups improved it to 100%.The yield of continuous mono-cropping group was the lowest(0.29kg/m2),rotation cropping group was the highest(0.61kg/m2),then was fermented silkworm excrement group(0.59kg/m2),lime group(0.51kg/m2),Vacterial manure group(0.50kg/m2)and silkworm excrement group(0.49kg/m2).From these date rotation was the most effective way to solve continuous cropping obstacles.While the farmland was limited,so rotation is difficult to implement and our fermented silkworm excrement undoubtedly a good solution which can significantly increase the survival rate and yield of continuous cropping Chrysanthemum morifolium.The present study shows that the continuous cropping can make the soil acidification.From the analysis of soil physical and chemical properties,we found that comparing with the soil pH,acid phosphatase and urease activity of continuous cropping group,the other five groups's were higher,they significantly improved the soil acidification caused by continuous cropping,which showed that there was a connection between soil physical and chemical properties and continuous cropping obstacle.and on the contrary,change soil physical and chemical properties such as pH,enzyme activity maybe can ease the continuous cropping obstacles.But,judging from the results of lime group,simply by adding lime to improve the soil pH worked,but not as good as using fermented silkworm excrement,so changing the pH is not the most efficient way.3?Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism(T-RFLP).The results show that enzyme Hae? is more appropriate than enzyme Hinfl because the number of TRFs digested by enzyme Hae? is more than that of enzyme Hinfl.In comparison of diversity,the endophytic bacterial communities's diversity index in group of cropping rotation and fertilizer is higher than continuous cropping which indicates that the addition of exogenous microorganism in soil could increase the diversity of plant endophyte.18 dominant species are selected,including 3 kinds of Firmicutes,4 kinds of Actinomycetes and 11 kinds of Proteobacteria.The results of dominant species comparison show that the number of dominant species in continuous cropping of Chrysanthemum morifolium is significantly less than the rotation group.Some dominant bacteria in rotation group and fertilizer group such as Arthrobacter,Streptomyces,Streptomyces,Flavobacterium and Mycobacterium are not found in the continuous cropping of Chrysanthemum morifolium group;Dominant species of fertilizer treatment group is similar with the rotation group,and the continuous cropping group's dominant species is more abundant.It indicates that these bacteria may be able to mitigate hindrance in continuous cropping,especially the Flavobacterium which can decompose the pathogenic fungi is worthy of further attention.Compared with leaves,there are more dominant species in roots and stems.The diversity of edophytic bacterial communities in continuous cropping of Chrysanthemum morifolium stays below than in the rotation of Chrysanthemum morifolium,and fertilizer treatment can increase the diversity of continuous cropping so that it could mitigate hindrance in continuous cropping.4?Rhizosphere soil samples was detected with T-RFLP.The results of Hinf I showed that compared with the Shannon index(3.60)of rotation cropping group,continuous cropping group decreased it to 3.31,while fertilization group was 3.57.The results of Hae III was 3.24,3.08,3.24,respectively.The bacterial diversity of continuous cropping group was obviously lower than rotation and fertilization group,shows that rotation and fertilization can significantly improve the level of diversity of rhizosphere bacteria which caused by Chrysanthemum morifolium continuous cropping.Through the dominant bacteria genera analysis it was found that there were 9 kinds of dominant bacteria genera in continuous cropping group,among them Clostridium,Phytoplasma and Xanthomonas were pathogenic bacteria;8 kinds of dominant bacteria genera in the Fertilization group,among them pathogenic bacteria Phytoplasma is missing,the only remaining pathogenic bacteria genera were Clostridium and Xanthomonas;8 kinds of dominant bacteria genera in the continuous cropping group,only Clostridium was detected.These results showed that fertilization and crop rotation processing will change the rhizosphere bacteria community structure mainly through reducing bacterial pathogens.Interesting founding was that Bacillus subtilis called biocontrol bacterium was detected in fertilization group.5?Using Miseq technology to detect hangzhou international bacterial community structure.Comparing with continuous cropping group,other 5 groups increased rhizosphere soil bacterial diversity.The main analysis results showed that the lime and the rotation group has similar bacterial community structure,the bacterial community structure of fermented silkworm excrement group,silkworm excrement group and bacterial manure group was similar.The advantage bacteria analysis showed that the content of Proteobacteria?Acidobacteria?Actinobacteria?TM7?Firmicutes?Planctomycetes?Bacteroidetes and Verrucomicrobia was up 90%.Compared with continuous cropping group,other 5groups increased the content of Acidobacteria and Verrucomicrobia.fermented silkworm excrement group had highest content of Actinobacteria and Planctomycetes comparing with other groups.Analysis of the genus advantage showed the 5 groups increased the content of beneficial bacteria,such as bio-control bacteria Bacillus and Burkholderia,azotobacter(Bradyrhizobium and Devosia),Chitinophaga and Sphingobium which can degrade allelochemicals.Conclusion Rotation cropping group,fermented silkworm excrement group,silkworm excrement group,bacterial manure group and lime group all had improved the continuous obstacle of Chrysanthemum morifolium,improved survival rate,yield,rhizosphere soil pH,acid phosphatase and urease activity,increased the rhizosphere bacteria diversity and promote the growth of beneficial bacteria such as bio-control bacteria Bacillus and Burkholderia;azotobacter(Bradyrhizobium?Devosia),Chitinophaga and Sphingobium. |
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