Diversity Of Endophytic Bacteria In Eucalyptus And Microecological Regulation Of Eucalypt Resistance To Bacterial Wilt

In order to reveal the biocontrol mechanism of endophytic bacteria against bacterial wilt in Eucalyptus,lay the foundation for the application of microecological regulation to control eucalypt bacterial wilt,and provide new research ideas for the control of the disease,the endophytic bacterial diversity in Eucalyptus was investigated from the perspective of microbiome.The endophytic bacteria in the aseptically cultured Eucalyptus tissue culture seedlings were systematically investigated using optical microscope,scanning electron microscope,and high-throughput sequencing technology.The effects of environmental and time factors on the endophytic bacterial community structure in Eucalyptus were analyzed using high-throughput sequencing technology to discuss its regulating potential.And preliminary study on the microecological regulation of Eucalyptus against bacterial wilt was carried out by the methods of combination of biocontrol bacteria and its compounding with fulvic acid.The main results were as below.(1)Ubiquitous presence of endophytic bacteria in Eucalyptus.The presence of endophytic bacteria was observed in the tissue culture seedlings of E.camaldulensis,E.trophylla,E.pellita,LH1,L22 and L39 of E.urophylla × E.tereticornis by using optical microscope at a magnification of 1000 times,most of which were coccus and elliptic shaped and measured 1?2 ?m.It was showed that the average numbers of endophytic bacteria in each observation field of Eucalyptus species and clones of E.urophylla × E.tereticornis were around 9.0 and 14.0,respectively.By using scanning electron microscope(SEM)at a magnification of 5000 times,the presence of endophytic bacteria was observed in roots,stems and leaves of the tissue culture seedlings of three Eucalyptus species,most of which were rod and elliptic shaped and measured 1?2?m.The endophytic bacteria in different organs of the tissue culture seedlings of three clones of E.urophylla × E.tereticornis were most rod shaped and measured about 2 ?m.Through counting under SEM at a magnification of 5000 times,the average numbers of endophytic bacteria in each observation field of Eucalyptus species and clones of E.urophylla× E.tereticornis were around 2.0 and 5.0,respectively.These results indicated the ubiquitous presence of endophytic bacteria in Eucalyptus.The quantities of endophytic bacteria in E.urophylla ×E.tereticornis were generally more than those in in vitro germinated seedlings of Eucalyptus species mentioned above.The endophytic bacteria in the tissue culture seedlings of E.camaldulensis,E.urophylla,E.pellita,E.urophylla × E.tereticornis and E.urophylla × E.grandis were detected and analyzed using high-throughput sequencing technology.The analysis of compositions and structures of the endophytic bacteria in the tissue culture seedlings of three Eucalyptus species and three clones of E.urophylla × E.tereticornis at different classification levels indicated that,the endophytic bacterial community structures were more similar among the tissue culture seedlings of Eucalyptus species and among the tissue culture seedlings of E.urophylla × E.tereticornis.However,the endophytic bacterial community structures in different tissue culture seedlings of E.urophylla × E.tereticornis were also certainly different.At genus level,the two dominant bacteria in the tissue culture seedlings of Eucalyptus species were Pseudomonas and Sphingobium.Rhodanobacter was the most dominant bacterium in the tissue culture seedlings of E.urophylla × E.tereticornis,and the two other dominant bacteria were Mycobacterium and Luteibacter,and there was 43.8%of Roseomonas in clone L39 of E.urophylla × E.tereticornis.Mycobacterium was the largest proportion in the tissue culture seedlings of E.urophylla ×E.grandis DH32-29,with the two other dominant bacteria,i.e.Pseudomonas and Sphingobium.PCA showed that the differences in the endophytic bacterial community structures among the different Eucalyptus species were small,but there was certainly difference among the different clones of E.urophylla × E.grandis,and the endophytic bacterial community structures between Eucalyptus species and E.urophylla × E.tereticornis varied greatly.Result based on alpha-diversity analysis showed that Shannnon indices of the endophytic bacterial community in Eucalyptus species were higher than those of all clones,while in the richness(Chaol),no regularity presented.Based on these results,the indigenous endophytic bacterial community structures of Eucalyptus species were stable,while the endophytic bacterial community structures were certainly different between different Eucalyptus clones and most different between Eucalyptus species and clones,and the the diversity of endophytic bacteria in Eucalyptus species was higher than that of clones.(2)Controllability of the endophytic bacterial community structure in Eucalyptus.Clone DH32-29 of E.urophylla × E.grandis was used to detect its roots under different environmental and growth time conditions to analyze the compositions and structures of endophytic bacteria at genus and species levels,alpha-diversity and beta-diversity of bacterial community using high-throughput sequencing technology.The results showed that the endophytic bacterial community structures between the roots of the 2-year-old Eucalyptus under different environment conditions were not much different.OTU number in the root of acclimatized seedling was the highest,and the proportion of unique OTUs reached 63.2%.However,the proportions of unique OTUs in other samples were all 30%or less.At the genus level,the structure of the endophytic bacteria in the root changed dramatically during the period from tissue culture seedlings to acclimatized seedlings,and the proportion of Afipia dropped sharply.It also changed significantly during the period from acclimatized seedlings to 1-year-old seedlings,and the proportion of Ochrobactrum increased significantly.However,it changed the least during the period from 1-year-old seedlings to 3-year-old seedlings.During the period from tissue culture seedlings to acclimatized seedlings,both Chao1 and Shannon indices of endophytic bacteria community in the root increased by about 1.7 times and 2.6 times,respectively,and both reached the highest values.The analysis results based on non-metric multidimensional scaling(NMDS)showed that the distance between groups of tissue culture seedlings and acclimatized seedlings was the longest,and the distance between the 1-year-old and 3-year-old Eucalyptus samples was the closest.Based on these results,the effect of environmental factor on the endophytic bacterial community structure and its increase of diversity in Eucalyptus were most significant during the stage of acclimatized seedlings,which was the best period for regulation.Three dominant bacterial genera and five dominant bacterial species were detected in all roots samples,and Ochrobactrum,Sphingomonas,Lysinibacillus sphaericus and Stenotrophomonas maltophilia had been reported as the biocontrol bacteria for plant diseases.(3)Microecological regulation of eucalypt resistance to bacterial wilt.The disease resistance of E.urophylla × E.grandis DH32-29 seedlings could be improved to a certain extent by the treatments including strains combination Pseudomonas fluorescens WCS417r and Bacillus subtilis CN181,fulvic acid,fulvic acid+WCS417r+ CN181.The disease incidences of Eucalyptus seedlings treated with WCS417r+CN181 treatment were lower than those of blank control and WCS417r treatments at each recording time.The disease incidences of Eucalyptus seedlings treated with fulvic acid treatment on the 7th,10th,and 15th days were lower than those of the control group and WCS417r+CN181 treatments,significantly lower than those of blank control and WCS417r treatments on the 7th and 10th days(P<0.05),and the incidences were reduced by 30.0%and 25.0%(7 d),45.0%and 35.0%(10 d),respectively.The disease incidences of Eucalyptus seedlings treated with fulvic acid+WCS417r+CN181 treatment were lower than those of the control group and WCS417r+CN181 treatments at each recording time,significantly lower than those of blank control and WCS417r treatments on the 7th and 10th days(P<0.05),and the incidence was reduced by 30.0%and 25.0%(7th d),50.0%and 40.0%(10th d),respectively.The disease incidences of Eucalyptus seedlings treated with fulvic acid+WCS417r+CN181 treatment on 5th and 10th days were higher than those treated with fulvic acid treatment,but the differences were not significant(P>0.05).The community structures of endophytic bacteria in E.urophylla × E.grandis DH32-29 on the 10th day treated with different combinations of biocontrol bacteria and fulvic acid were detected using high-throughput sequencing technology to explore the relationship between the dynamic changes of endophytic bacteria and the disease resistance of Eucalyptus from the perspective of microecology.The OTU number,PCA and bacterial composition analysis results showed that the bacterial structures were very different in the tissue culture seedling sample and other samples,with the highest similarity of the bacterial structures in the treatment group samples.After the tissue culture seedlings were transferred from aseptic condition to the natural environment,the endophytic bacterial community structure changed a lot,and the bacterial community structures in treatment group samples were most similar.The abundances of detectable endophytic bacteria in the tissue culture seedlings were very low but were higher in other samples.Compared with the samples of tissue culture seedlings and sterile distilled water treatment,the abundances of Pseudomonas and Sphingobium in the positive control group samples treated with single biocontrol bacterium and the treatment group samples treated with different combinations were increased greatly.The abundances of Flavobacterium in the samples treated by CN181,WCS417r+CN181,fulvic acid,fulvic acid+WCS417r+CN181 with better effects on improving disease resistance of Eucalyptus was higher,and as the disease resistance increased,the proportion of Flavobacterium increased by 11.1%,12.6%,15.7%and.7.9%,respectively.Alpha-diversity analysis results showed that Chao1 indices of the endophytic bacterial community in these four treated samples were higher than those in other samples,and both Chao1 and Shannon indices of these four samples were significantly higher than those of samples of tissue culture seedlings and WCS417r treatment(P<0.05).These results indicated that the ability of eucalypt resistance to bacterial wilt could be improve by the endophytic bacterial community structure after microecological regulation,and the increase of the proportion of Flavobacterium in Eucalyptus might be the key of microecological regulation.