| Peach gummosis is one of the most serious diseases on peach trees in China,and such disease is mainly caused by the fungal pathogen Botryosphaeria dothidea,B.rhodina and B.obtuse.At present,the prevention and control strategy for the peach gummosis relies on chemical control and supplemented with agricultural means.However,the use of chemical pesticides not only induces drug resistance for pathogens,but also causes environmental pollution and endangering human health.Biological control is widely concerned because of its environmental protection,safety and reliability.In this experiment,an endophytic bacteria was isolated and screened from the root tissues of peach tree and it has antagonistic effect on the pathogen of peach gummosis.The bacteria were identified by morphological observation and molecular identification.The colonization of the strains in plant was studied by GFP markers.In addition,the biocontrol mechanism of the strain was preliminarily discussed.The main results are as follows:1.The total of 129 strains of endophytic bacteria were isolated from the root and leaf parts of the peach tree,of which 20 were antagonistic to the pathogen B.dothidea XNHG-241 of peach gummosis.One strain,HAR3 which had better antagonism was identified.The phylogenetic tree was constructed by 16 S rRNA and gyrB gene sequence and combined with morphological observation,the strain HAR3 was identified as Bacillus amyloliquefaciens.2.The plasmid pUBXCP with egfp marker was constructed and then was transferred into the B.amyloliquefaciens by conjugational transfer approach.The obtained strain HAR3-GFP could express strong fluorescence and stable heredity.Importantly,the inhibitory effect of strain HAR3-GFP on B.dothidea XNHG-241 was comparable to wild type HAR3.3.The antifungal spectrum test showed that HAR3 not only had antagonism against the peach gummosis-inducing pathogen B.dothidea,B.rhodina and B.obtuse,but also had antagonistic effect on many other pathogens,such as Botrytis cinerea,Monilinia fructicola,Colletotrichum higginsianum,Penicillium digitatum and Geotrichum citri-aurantii.Interestingly the strain HAR3 had no antagonistic effect on Rhizopus stolonifer.4.The plant was irrigated with HAR3-GFP suspension,and then roots of tomato and Arabidopsis seedlings were sliced and photographed under the confocal laser scanning microscope.After inoculation 1 d and 3 d,large numbers of HAR3-GFP were colonized on the root and root hairs.After 5 d,only a few colonies were observed.By using tissue separation and dilution plating,after 10 d,30 d and 100 d inoculation,HAR3-GFP could be separated from the roots and leaves of peach trees,indicating that HAR3-GFP could be colonized in the peach tree and could be extended to the stem and leaf through the root.5.HAR3 suspension was sprayed on peach branches,where the area was immediately inoculated with the pathogen B.rhodina JMB-122.After 5 d inoculation,the lesion of treatment group was 4.36 mm,approximately 32.8% reductions compared to the control.For the gum flow,there were no gum release in the treatment group,while the gum flow rate of control was about 75%.On the other hand,spraying HAR3 suspension on the branch after 24 h,we then inoculated B.dothidea.In the control group,the lesion reached 5.16 mm at 5 d,and the rate of flow gum reached 25%,In details,in the treatment group the lesion was 0.65 mm,about 87.4% reductions,and no no gum flow occurred at 5 d.6.The fermented crude extracts were prepared with trichloromethan:methanol(65:15)from bacterial fermented supernatants of the strain HAR3.The growth of B.dothidea XNHG-241 could be completely inhibited when 0.5625% of the fermentated crude extracts were added into the medium.Meanwhile,eight different lipopeptide synthesis genes were amplified from HAR3,including bamD gene for bacillomycin D synthesis,bacD,bacAB genes for bacilysin synthesis,and ituD,ituA,ipa 14,ituC genes for iturin synthesis. |
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