| ObjectiveAquilaria Sinensis(Lour.)Gilg,known as soil aloes or daughter ince nse,is the plant of the genus Daphne,the Pharmacopoeia of 2010 as the o nly source of aromatic herbs and aloes in China,is classified as the nat ional two-level endangered plant protection.However,the traditional met hod of Aquilaria in natural conditions has long time of fragrant knot in medicine,there are more than more than 160 kinds of prescriptions have t heir compatibility with the digestive system,cardio-cerebrovascular,tum or and other diseases,but also chemical production,precious spices prod uction or precious handicraft production of raw materials,resulting in t he market demand for aloes,and have to rely on imports,expensive,marke t gap.Early in the early 20th century,foreign scholars deduced that the formation of aloes was related to the fungal infection,and the study of endophytic fungi in the domestic and international wood was concentrated in the study of endophytic fungus diversity,species separation,identif ication,active ingredient research and possible physiological function r esearch.It was discovered in 2013 that there are abundant endophytic bac teria in the genus Aloes,which may be related to plant aroma.At present,most of them are based on traditional culture methods,and few studies h ave been reported on the relationship between Woody endophytic bacteria.Therefore,the traditional separation method was used to study the specie s and distribution of endophytic fungi in white Woody wood,and to provid e solid bacteria resources for subsequent experiments,objective to inves tigate the distribution of endophytic bacteria(including endophytic bact erium and endophytic fungi)in different fragrant wood and different arom atic sites,and to study the endophytic bacteria and endophytic fungi in white Woody by using Dgge(polymerase chain reaction-denatured gradient g el electrophoresis).To investigate the changes of endophytic bacteria an d the community structure in different parts of white woody culture two m ethods were used to find out the similarities and differences of the spec ies and distribution of endophytic fungi under two methods,and to elucid ate the role of endophytic bacteria in Aquilaria junction incense by usin g the distribution law of discovery.MethodThe study of endophytic bacteria and endophytic fungi in white Woody Wood was studied by means of separating culture and dgge molecular Biotechnology.The specific experimental methods are as follows:(1)Sample collection and processingCollection of Dongguan Dayushan Shang aloes Production,collection of injuries 1 years,2 years,3 years,4 years,5-year Aquilaria samples(including white wood parts,rotten wood parts,incense parts),with each sampling before disinfection of the growth cone to obtain the Aquilaria target part,immediately into the sterilization bag,refrigerated storage transport.The 5-point Aquilaria sample repeats 3 samples,totaling 15 white woody samples(including the white wood portion 15,the rotten wood portion 15,the fragrant portion 15).(2)Sample surface sterilizationThe sample in the aseptic bag is taken out in the super net and rinsed 1 times with sterile water,respectively in the sterile culture dish,completes the mark,70%ethanol soaks 2min,and then move into a petri dish containing 0.9%sodium hypochlorite solution,soak 5min,rinse more than 3 times with sterile water,transfer to the sodium bicarbonate solution 15min,which has been filtered and 10%,and finally rinse more than 3 times with sterile water,and sterilize the sample surface.(3)Traditional isolation culture and dgge analysis of endophytic bacteria strains and their distribution in white Woody woodDiscussion on endophytic bacteria flora and distribution based on traditional culture method:The sample was separated and purified by tissue-block method,and the samples were treated in aseptic operation environment.The outstanding fragrant part and the rot layer cut to 1 cm or so length,respectively put into the separation plate PDA medium(potato glucose agar)+ streptomycin(50u Ml)after lightly presses,in the incubator incubator culture observation,several times the board inoculation to purify the bacterium,the colony photographed,microscopic photograph and so on.The strains obtained from isolation and culture were identified by colony identification,microscopic identification and molecular identification.Endophytic bacteria are preserved in 70 Shan by 25%Glycerin,and endophytic fungi are stored in liquid paraffin wax at normal or low temperature.The diversity of the Simpsons Diversity Index was used to reflect the genotype diversity of the endophytic community in 3 locations at the same time and the characteristics of the colony structure at 3 sites at different time.The Simpson index formula D= 1-?(Ni(Ni-1))/(N(N-1)),and Ni is the number of strains in a sample,and N is the number of all strains in the sample.And the distribution regularity of endophytic bacteria flora is vividly reflected by the Venn graph and line graph.Discussion on the bacterial flora and distribution of endophytic bacteria based on molecular biotechnology Dgge:The total DNA of the aromatic part and the white wood part of the white woody were extracted respectively,and the bacterial 16S rDNA general primers were amplified,and the modified gradient gel electrophoresis was used to understand the non cultured endophytic bacteria strains and to compare the structure differences of the endophytic bacteria.The genotype diversity(Genotypic Diversity)Characteristics of the bacterial community before and after Aquilaria,the relative degree(pi)of the genotype was measured by quantity-one software.With genotype abundance(Genotypic Richness,S),Simpson index(Simpsons Diversity Index,D)and Pielou evenness index(Pielou Evenness Index,J).D=-E Pi'2 J=H'/HmaxPi=ni/N,ni is the degree of the section I bands,N is the total degree of all bands in the sample,and Hmax=lnS,S is the total number of strips in the sample.Statistically,single factor variance analysis(ANOVA)was used to determine whether there were significant differences in genotype diversity of endophytic bacteria in Aquilaria group and in the No.ANOVA is implemented by SPSS19.0 statistical software.Results(1)Analysis of endophytic strains in white woody wood by traditional method of isolation and culture:Endophytic bacteria:Isolated from aquilaria samples of about 27 endo phytic bacterial strains,of which Bacillus(Bacillus sp.)and Burkholder ia(Burkholder sp.)are predominant strains of these endophytic bacteria,3 different parts have them,the total number of them in different acqui sition time also occupies the main position,the Burkholderia(Burkholder genus)mainly in the Aquilaria part,Bacillus(Bacillus sp.)is mainly i n the Aquilaria junction.In three sites,the diversity index was DJ>DF>DB,the diversity of the endophytic fungi was the most,the diversity of the endophytic fungi in the white wood was the fewest,and the community str ucture of the endophytic bacteria in the environment of the white Woody w as different.Under the same site,endophytic bacteria in different sampl ing time of endogenous bacterial community genotype diversity,the white Wood Part diversity index presents DB1>DB4>DB5>DB2>DB3,that is,aquilaria part of the diversity of endophytic bacteria in the first year of the ric hest,the lowest diversity in the last year;DF1 =DF2>DF3 =DF4=DF5,which i s the richest in the first and second year of the Humic species,the thir d year,fourth year and fifth year were the least endophytic bacteria;Th e diversity index was DJ2>DJ3>DJ4>DJ1>DJ5,and the second year endophytic bacteria were still abundant,and the endophytic bacteria were the least in five years.Endophytic fungi:About 46 endophytic fungal strains were isolated from aquilaria samples,among them,Penicillium(Penicillium sp.),Lasiodiplodia(Cacao),Trichoderma(Trichoderma sp.),Aspergillus(Aspergillus sp.),Fusarium(Fusarium sp.)are the predominant bacteria of these bacteria,and 3 different parts have them.Penicillium(Penicillium sp.)and Aspergillus(Aspergillus sp.)are mainly in the Aquilaria white-wood part,Trichoderma(Trichoderma sp.),Fusarium(Fusarium sp.)mainly in the Aquilaria part of the rot.The gene-type Simpson index of the three-site endophytic fungi community is DB>DJ>DF.The diversity of endophytic fungi flora in the Aquilaria white wood is the richest,and the diversity of the community structure of the endophytic fungi in the different parts of the environment will be different.Under the same site,the distribution and significance of endophytic fungi under different harvesting time were evaluated by Simpson Index.DB1>DB4>DB5>DB3>DB2,and the diversity of endophytic fungal flora in the Aquilaria part was the richest in the first year.The diversity of the second year was lowest;The Simpson index was DF4>DF5>DF3>DF1 =DF2,the third year,fourth year and fifth year endophytic fungi were richer than those in the first and second years of the tree.DJ3 =DJ5>DJ2>DJ1>DJ4,the most abundant endophytic fungus in the third year and five years,the lowest endophytic fungus in the fourth year.(2)Analysis of endophytic bacteria strains in white woody wood by Dgge technique:Endophytic bacteria:The Dgge Atlas cluster shows that most of the samples have a higher bacterial group similarity,and the bacterial state similarity between the samples is higher.Diversity analysis showed that the richness of aroma and aroma and the difference of Simpson index in the same plant were statistically significant,indicating that the species and number of endophytic bacteria were changed before and after agarwood formation,and the aromatic group was higher than that in the open group.Dgge the blast of the stripe sequence,the result is that the group consists of Flavobacteri ia Bacterium(Bacillus thuringiensis),Streptomyces sp.(Streptomyces sp.),etc about 10 endophytic bacterial flora;the Nesterenkonia sp(moderatealine genus)and Sphingobacterium multivorum(Sphingobacterium multivorum)endophytic bacteria.Among them,the Sphingobacterium multivorum in the Aquilaria white-wood part,the fragrant group is more abundant and diverse endophytic bacterial flora than the fragrant group samples.Endophytic fungi:The Dgge Atlas shows the number of bands,the location of the band,there is a difference between the group and the Incense group,on the whole,the number of knot incense group more than the outstanding group.The clustering of the samples showed that the similarity of the bacteria group states was higher together,and the similarity of the bacteria in the samples was higher.Diversity analysis showed that the richness of aroma and aroma in the same plant and Shannon Diversity index were significantly higher than those in the Open Aromatic Group(P<0.01),and the species of endophytic fungi increased obviously after fragrant.Dgge The blast ratio of the collected stripe sequence,the group of Aspergillus(Aspergillus sp.),Penicilliun(Penicillium sp.)and so on,the group has Corticium,Neurospora,etc.about 18 endophytic fungi.This part of the data is the result of experiments previously studied in this laboratory.Conclusion:(1)The endophytic bacteria in wood incense were obtained by means of culture method and non cultured molecular biological methods,including endogenous and non cultured bacterial flora and their distribution regularity.The diversity of endophytic bacterial flora at different time points was different,and the difference was correlated.The two methods were compared and found that the Bacillus(Bacillus sp.)and Burkholderia(Burkholder sp.)were common dominant strains,and the two strains could be the key bacteria in the Aquilaria junction incense.The results of comparison showed that the diversity analysis of the two methods was consistent,that is,the diversity of endophytic bacterial flora of Aquilaria Junction was higher than that of white Woody,and there was a definite difference between aquilaria and aroma.(2)The endophytic fungi in wood incense,through the methods of culture and the methods of non cultured molecular biology,obtained more information about endophytic fungi,including endogenous and non cultured fungal flora and their distribution.The diversity of endophytic fungi in different time was not correlated,and the variation of endogenous bacteria was more complicated than that of endophytic bacterium.The two methods were compared and found that there were common strains Aspergillus(Aspergillus sp.)and Penicilliu(Penicillium sp.),and the common strains were Trichoderma(Trichoderma sp.),which belonged to the dominant bacteria in the study of the cultivated and non cultured methods.The comparison results also found that the results of the diversity analysis of the two methods were inconsistent,that is,the Dgge diversity analysis results showed that the richness of endophytic fungi community was significantly higher than that in the aromatic group,and the diversity of the cultured methods was lower than that of the Aquilaria group.The difference of the two results reflects that the white woody endophytic fungi may have complex regularity in the process of aroma,not simple linear relation.The synthesis shows the rich and complex structure of endophytic fungal flora. |
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