| The120endopytic bacterial isolates were isolated from surface-sterilized leaves ofGinkgo biloba, which collected from Hangzhou, Zhejiang province, Zhengzhou, Henanprovince, Taian, Shandong province and Fuzhou Fujian province. The21bacterialisolates isolated from about50-year-old Ginkgo biloba leaves(Shandong province),62bacterial isolates isolated from about30-year-old Ginkgo biloba leaves(Henan province),17bacterial isolates isolated from about10-year-old Ginkgo biloba leaves(Fujianprovince),20bacterial isolates isolated from about100-year-old Ginkgo bilobaleaves(Zhejiang province).The results indicated that many endopytic bacteria existed inGinkgo biloba leaves and the endopytic bacterial populations had a significant differencein Ginkgo biloba leaves of different tree ages, that is about50-year-old＞about30-year-old＞about10-year-old＞about100-year-old. Using16S rDNA PCR-RFLP、ERIC-PCR and DNA gyrase B subunit （gyrB） sequence analysis, genetic diversity ofthe endophytic bacteria within Ginkgo biloba leaves were explored. The results showedthat the endopytic bacteria had a rich population diversity in Ginkgo biloba leaves.The120strains obtained42restriction endonuclease types based on the16S rDNAPCR-RFLP analysis.Further, the64isolates having predominant genotype were dividedinto53ERIC-PCR fingerprintings.The results showed the genetic diversity ofpredominant endophytic strains was high. The gyrB sequences of representative strains ofeach RFLP types were tested and the phylogenic trees were constructed.The resultsrevealed that these isolates belonged to26species of9different genera, and endopyticbacterial isolates belonged to genera of Pseudomonas and Bacillus that were thepredominant genera.Based on screening in vitro, the35bacterial isolates among120strains showedobvious inhibition ability on the mycelia of Phytophthora capsici.Especially, the strainsZy44、Fy11、Hy7、Hy14、Zy25、Zy45、Zy55and Sy15showed strong inhibitory activitywith more than10mm inhibition zones on plates. The8isolates showed differentinhibiting effect on plant pathogens in the dual plate assays. Further, the8isolates werechosen to control fruit phytophthora blight of pepper. The results showed that all thetested isolates could control the disease with different extent, and the isolates, Zy44、Fy11、Hy7、Hy14and Zy25showed significant biocontrol activity to the disease withmore than70%control effect in6d after pathogen challenge. The5isolates wereevaluated their biocontrol effects on seedling phytophthora blight of pepper ingreenhouse experiments. The isolates, Zy44and Fy11, were found to have betterbiocontrol efficacy with more than50%control effect in20d after pathogen challenge. Based on the morphological, physiological, biochemical characteristics,16S rDNA andgyrB sequence analysis, the strains Zy44、Fy11、Hy7and Zy25were identified as Bacillusamyloliquefaciens.The3biocontrol strains, Fy11、Hy7and Zy44, were tested in combinations forbiological control against fruit and seedling phytophthora blight of pepper. The mixturesof Fy11and Zy44showed significant reductions in disease severity on fruits andseedlings phytophthora blight of pepper after pathogen challenge, comparing with othercombinations and a single strain The control effects were76.3%and71.3%respectively,.Further, the3strains were evaluated their growth promotion effects on pepper seedling ingreenhouse experiments. The results indicated that the endophytic strains, Fy11、Hy7andZy44, were also effective for promoted the growth of pepper seedlings with43.65%、39.53%and46.93%growth-promoting efficacy after treated in30days,respectively. Thebiocontrol strains, Fy11and Zy44，were labeled with green fluorescent protein gene toinvestigate the colonization of the bacteria in the pepper. The2strains were able tocolonize in the internal tissues of the pepper and transport into stem and leave from root.The colonization density of Fy11was higher than that of Zy44in the pepper tissuethroughout the duration of the experiment, especially stem and leaves of the pepper. Theendophytic colonization of mixtures of two strains was not significant difference incontrast to a single strain. Direct antagonism of antifungal compounds produced byendophytic bacteria was investigated.The cell-free filtrate and lipopetides produced bythe strain Zy44was able to suppress effectively seedling phytophthora blight of pepper,and the lipopetides compounds were better than cell-free filtrate, but direct antagonism ofculture filtrate and lipopetides produced by Fy11was not obvious. It was observed bylight microscopy that the lipopetides compounds produced by the strains Zy44couldinduce alternations on hyphae morphology and reducing the sporangia production ofP.capsici. The induced systemic resistance (ISR) mediated by Fy11and Zy44on pepperwas further analysed using priming effect of the defense-related genes after pathogenchallenge. qRT-PCR analyses revealed that the expression of the defense-related geneswas primed by the application of strain Fy11, especially, the expression of the CaPR4(Capsicum annuum pathogenesis-protein4) gene, but the expression levels ofdefense-related genes were not obvious different in plants treated with Zy44-, SA-, andthe pathogen.The biocontrol function gene fragments of bmyB, fenD, bioA, srfAA, srfAB, bioA,yngG and yndJ were amplified with specific primers from genomic DNA of the twobiocontrol strains, Zy25and Hy7, which indicated that the two strains could producelipopetide metabolites. The lipopetides compounds produced by Zy25and Hy7exhibitedinhibitory activity on mycelium growth of some plant pathogens detected. The mycelia of plant pathongens treated with lipopetides compounds appeared distortion，protoplasmagglomerated and stopped growing，which were observed by light microscopy. Thelipopetides compounds produced by Zy25and Hy7were evaluated their biocontroleffects on fruit of pepper phytophthora blight and tomato gray mould.The results showedthat lipopetides compounds produced by Zy25and Hy7were able to reduce effectivelydisease sevisity.cDNA-AFLP technique was used to conduct transcription profiling of pepperseedlings across five sampling time points after inoculation with the strain Fy11. Theresult showed that the total18620transcript derived fragments (TDFs) were obtained byamplification with256primer pairs, among which353(1.89%) displayed alteredexpression patterns after inoculation. Two hundred and fifty seven differentiallyexpressed TDFs produced reliable sequences after cloning and sequencing. Two hundredand twenty nine expressed sequence tags (ESTs) of unigenes were obtained afterassembling, of which144showed up-regulated and85down-regulated. Blastx analysesand functional annotations were then performed and the results revealed that the156ESTs had predicted gene products mainly implicated in energy (10.92%), metabolism anddisease/defense (each accounted for8.73%), signal transduction (7.42%),transporter(6.99%), cell structure(6.55%), transcription(5.68%), cell growth (3.93%),protein destination and storage(3.49%), protein synthesis (2.18%), secondary metabolismand intracellur traffic (each accounted for1.75%). The65EST（s28.38%of the sequencedtotal229ESTs) had no match to known genes, the8ESTs(3.49%of the sequenced total229ESTs) were highly homologous to unknown functional proteins. Ten differentialgenes related to disease/defense, transcription and signal transduction were chosen forfurther qRT-PCR expression patterns to evaluate the validation of cDNA-AFLP. Theresults showed that these genes expression patterns confirmed the cDNA-AFLPprofiles. |
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