Research On The Community Structure Of Mulberry Endophytic And Rhizosphere Fungi And Their Improvement Of Stress Resistance In The Hydro-fluctuation Belt Of The Three Gorges Reservoir Area

The Three Gorges Reservoir is periodically adjusted to make the water level fluctuate from 145 m(summer)to 175 m(winter)in an unseasonal manner.Therefore,the periodic rise and fall of the water level in the reservoir creates a 30-meter hydro-fluctuation belt zone,which resulting in biodiversity loss and soil erosion in the hydro-fluctuation belt.Mulberry(Morus L.)is an indigenous tree native to the reservoir area,which has the advantage of drought and flood resistance,water and soil conservation and so on.Therefore,it can be used as a dominant species to repair the ecological environment in the hydro-fluctuation belt.However,the ecological environment in the hydro-fluctuation belt is complex,which leads to the low survival rate of mulberry newly planted in the reservoir area.Therefore,how to improve the survival rate of the newly planted mulberry and promote the overall growth of mulberry is an urgent problem to be solved in improving the ecological environment of the hydro-fluctuation belt in the reservoir area by using mulberry.At the early stage,the project team observed that there were obvious differences in mulberry in the hydro-fluctuation belt of Longjiao Town,Yunyang County,the Three Gorges Reservoir area.Mulberry cultivated for ecological restoration were partly luxuriant(GM).Some without disease,but the stems were short and thin(PM).The surviving wild mulberry,which existed in the hydro-fluctuation belt before the completion of the Three Gorges dam had developed overall roots and stout stems(WM).This study selected the topic based on the observation of mulberry growth status in the hydro-fluctuation belt of the Three Gorges Reservoir area,and the community structure of endophytic and rhizosphere fungi of mulberry with different growth potential in the hydro-fluctuation belt of the reservoir area was investigated with metagenomics and culture method.On the basis of isolated and obtained rhizosphere and endophytic fungi,strains with potential of promote growth and resist stress were screened and their basic biological characteristics were studied.Based on the experiment of promote seedling growth by potted cultivation,the tolerance of seedling to drought was further explored,and the purpose of this study was to provide a strategy for recovered the vegetation and improved the ecological environment in the the hydro-fluctuation belt by using the endophytic and rhizosphere fungi of mulberry.1.Community structure analysis of mulberry rhizosphere and endophytic fungi with different growth status in the hydro-fluctuation beltHigh-throughput sequencing based on Illumina MiSeq was used to analyze the endophytic and rhizosphere fungal community structures of mulberry in the Yunyang segment of the three Gorges Reservoir area(May 2016 and May 2018).It was found that the distribution characteristics of mulberry related fungi were consistent as a whole.The ?-diversity showed that: the abundance of good growth cultivated mulberry was higher than that of wild mulberry,and the abundance of mulberry rhizosphere was higher than that of mulberry endophytic fungi;The fungi diversity of cultivated mulberry was higher than that of wild mulberry,and the fungi diversity of mulberry stem was higher than that of mulberry root and rhizosphere.The results of the analysis of ?-diversity based on fungal community showed that the sampling location(root,stem and rhizosphere soil)and the growth status of mulberry trees all had an effect on the distribution of endophytic and rhizosphere fungi,and at the same sampling location,the related fungal community of mulberry can also be separated from each other according to their growth.And the endophytic fungi of good growth and wild mulberry were more similar.Analysis of fungal community structure found that: At the phylum level,Ascomycota and Basidiomycota were the dominant fungi in wild mulberry(>75%),while Ascomycota and Mortierellomycota were the dominant fungi in cultivation mulberry(>80%).The endophytic fungi(root and stem)of mulberry were all dominated by Ascomycota(>75%),but Ascomycota,Basidiomycota and Mortierellomycota were the dominant fungi in rhizosphere of mulberry(>90%).At the class level,Agaricomycetes was the dominant fungi in wild mulberry(>19%),while Sordariomycetes was the dominant fungi in cultivation mulberry(>18%).The endophytic fungi(root and stem)of mulberry were all dominated by Sordariomycetes ?Dothideomycetes and Leotiomycetes(>40%),but Agaricomycetes and Sordariomycetes were the dominant fungi in rhizosphere of mulberry(>20%).From the traditional separation culture method,a total of 247 strains were isolated from mulberry in May 2018,belonging to 3 phyla,7 classes and 42 genera.Culture method and non-culture method showed that the structure of endophytic and rhizosphere fungi community of mulberry was similar.In hydro-fluctuation belt,Ascomycota,Basidiomycota and Mortierellaomycota were the dominant flora at the phylum level in mulberry trees.Sordariomycetes,Dothideomycetes and Leotiomycetes were the dominant flora at the class level in mulberry trees.2.Screening,identification and biological characteristics of mulberry rhizosphere and endophytic fungi with potential for plant growth promoting and stress resistanceOn the basis of the rhizosphere soil and endophytes fungi obtained from the mulberry samples in May 2018,by detecting the ability of the strains isolated to dissolve phosphorus,produce indole acetic acid(IAA),use 1-amino cyclopropane-1-carboxylic acid ester(ACC),the strains with good anti-stress and pro-growth potential and non-antagonistic ability were screened from the growing mulberry(WM,GM)related fungi(endophytic fungus GR19,GR21 and GS1,rhizosphere soil fungus GRs12)to provide strain resources for subsequent studies.Based on morphological observation and 18 S rDNA and ITS sequence analysis showed that placing GR21,GR19,GS1 and GRs12 strains identified as Trichoderma sp.,Penicillium sp.,Talaromyces sp.and Pseudeurotium sp.,respectively.The results of tolerance to salt,pH and temperature showed that Penicillium sp.GR19,Talaromyces sp.GS1 and Pseudeurotium sp.GRs12 strains could grow in PDB containing 8% NaCl,and Trichoderma sp.GR21 strain could grow in PDB containing 7% NaCl.All four strains were able to grow at pH 3-12.GR19,Trichoderma sp.GR21 and Talaromyces sp.GS1 strain can grow vigorously at 10?C to 35?C,while Pseudeurotium sp.GRs12 strain did not grow at 35?C.The results of functional enzyme production and pro-generative potential test showed that Penicillium sp.GR19,Talaromyces sp.GS1,Pseudeurotium sp.GRs12 strains with pectinase activity,Penicillium sp.GR19,Talaromyces sp.GS1,Trichoderma sp.GR21 strains with cellulose enzyme activity,Penicillium sp.GR19,Talaromyces sp.GS1 strains protease activity.Pseudeurotium sp.GRs12 and Trichoderma sp.GR21 strains could produce iron carriers to absorb iron ions from the surrounding environment for use by plants.The dissolved phosphates of Talaromyces sp.GS1,Pseudeurotium sp.GRs12 and Penicillium sp.GR19 strains were 0.699 mg/ml,0.431 mg/ml and 0.032 mg/ml,respectively.The produce IAA content of Pseudeurotium sp.GRs12 and Trichoderma sp.GR21 strains were 1.220 mg/mL and 0.062 mg/mL,respectively.ACC deaminase activities of Trichoderma sp.GR21,Pseudeurotium sp.GRs12 and Talaromyces sp.GS1 strains were 0.31 U/mg,0.86 U/mg and 0.45 U/mg,respectively.The results showed that the screening of strains with good resistance to adverse environment capacity,some strains with good growth potential(IAA,dissolved phosphate),part of the strains have good potential of resistance to adverse environment(can secrete ACC deaminase),and no antagonism between these strains,can build compound fungi agent,to promote the growth of mulberry sapling,and promotion of the new plant mulberry survivability strategy.3.Study on promoting the growth of mulberry seedlings and improving its stress resistance by probioticsProbiotics fungi suspension of different concentration gradient had no significant effect on mulberry seed germination,while the Talaromyces sp.GS1 and Pseudeurotium sp.GRs12 strains original solution,Penicillium sp.GR19 and Trichoderma sp.GR21 strains diluted by 10-fold,and four strains mixed by 100-fold(Mixture 100X)was increased the biomass of mulberry seedlings 263.03%,292.31%,258.43%,281.12% and 287.34%,respectively.Besides,the physical and chemical properties of potting soil and the composition of microbial community were analyzed.The results of physicochemical property showed that fungi can change the physical and chemical properties of rhizosphere soil(Iron ion,organic matter,available phosphorus and total phosphorus)to promote the absorption of nutrients by host plants.Meanwhile,probiotics changed the fungal community structure in the soil of potted mulberry seedlings,while the probiotics had no effect on the bacterial community structure.According to the experimental results of promoting the growth of mulberry seedlings,the compound fungi agent was constructed by the mixture of 100-fold diluted solution(Mixture 100X)and the mixture optimal concentration of single strain(Mixture optimal)to evaluate its effect on drought resistance of potted mulberry seedlings.The results showed that mixture probiotic fungus inoculation could improve the anti-stress potential of mulberry seedlings.The detection was conducted at the 5-days of drought stress,the superoxide dismutase activity,soluble sugar and chlorophyll contents of mulberry seedlings in the Mixture 100 X treatment group were increased by 267.93%,34.35% and 16.53%,respectively,while the contents of soluble sugar,leaf nitrogen and chlorophyll in the Mixture optimal treatment group were increased by 176.47%,21.18% and 12.31%,respectively.The recovery of mulberry seedlings under drought stress at 1 d and 3 d treated with the two compound fungi were similar,and there was no significant difference between the two groups and the control group under drought stress.Under drought stress at 5 d,the recovery of mulberry seedling with Mixture Optimal treatment was better than that of the control group,and the degree of leaf drop was slower.Meanwhile,the two compound fungi agent could increase the biomass of mulberry seedlings under drought stress and change the community structure of soil fungi under drought stress,so as to improve the resistance of host mulberry seedlings under drought stress.Probiotics can promote the growth of mulberry seedlings and improve their stress tolerance potential by inducing host plants to produce anti-stress enzymes and osmotic protective substances,changed the physicochemical property of soil,and affected the structure and composition of soil microbial community.In summary,the results of this study indicated that the sampling location(root,stem and rhizosphere soil)and the growth status of mulberry trees all had an effect on the distribution of endophytic and rhizosphere fungi,and at the same sampling location,the related fungal community of mulberry can also be separated from each other according to their growth.And the endophytic fungi of good growth and wild mulberry were more similar.Probiotic fungi Trichoderma sp.GR21,Penicillium sp.GR19,Pseudeurotium sp.GRs12 and Talaromyces sp.GS1 screened in this study could promote the growth of mulberry seedlings,and could improve the stress resistance of mulberry seedings.This study provided a good source of fungi and a new strategy for the later development of mixture agents and the subsequent use of mulberry for vegetation restoration in the three gorges reservoir area.