Isolation And Identification,Fermentation Conditions Of Alocasia Endophyte TNX-1 And Study On Antifungal Substances Of The Fermentation Liquid

Fungal diseases of longan fruits is the main reason for corruption on post-harvest storage.Lasiodiplodia theobromae(Pat.)Griff&Maubl and Phomopsis longanae Chi.are the main latent pathogenic fungi on storage.Endophytic bacterium TNX-1 isolated from alocasia plant by the study has a strongly inhibition on them.Therefore,this thesis identified on the TNX-1,then study on its ferment conditions,and the extraction method,physicochemical properties and mechanism of its antifungal target product.The results were as follows:1.The results of identification:Colonies of TNX-1 isolated from alocasia plant were yellowish,rough and opaque,and its surface was dry and jagged.It formed a thin film when cultured in liquid medium.It was gram-positive,rod-shaped stains that single arranged or arranged in a pair or in a chain,central spores oval.Its morphological characteristics,physiological and biochemical analyses were similar to Bacillus subtilis and Bacillus amyloliquefaciens.From the analysis of the 16S rDNA gen sequence,this strain was more 99%homology with Bacillus.In the phylogenetic tree,the TNX-1 and Bacillus amyloliquefaciens(HM989844)were in the same branch and the credibility is up to 99%.It could not be amplified a single band of 1300 bp when using specific primers Bsu of Bacillus subtilis,but it could be amplified a single band of 1300 bp when using specific primers of Bam Bacillus amyloliquefaciens,and it could be amplified a single band about 736 bp when using specific primers of Multiplex.So TNX-1 was identified as Bacillus amyloliquefaciens and named as Bacillus amyloliquefaciens TNX-1.2.Fermentation conditions of Bacillus amyloliquefaciens TNX-1:The single factors including temperature,carbon sources,nitrogen sources and initial pH were studied for producing antifungal target product by TNX-1,and then design and orthogonal tests and verification tests,and finally add the metal ion to optimize,the optimum fermentation conditions for medium formula:3%of soybean meal,0.2%of ammonium acetate,0.1%KZHPO4,and KCl 0.050%,MgS04·7H2O 0.05%,0.001%of FeS04 and mannitol 0.2%,not adjusted to pH;temperature:32?35?;inoculum size:5 percent;ventilation:75 mL/150 mL;speed:170 rpm;incubation time:76 hours.3.Extraction methods of Bacillus amyloliquefaciens TNX-1's fermentation antifungal substances:The methods including organic solvent extraction,ammonium sulfate fractionation,macroporous resin adsorption and acid precipitation were used to extract antifungal substances from the fermentation broth.It is shown that the extraction efficiency of ammonium sulfate fractionation was higher than organic solvent extraction and macroporous resin adsorption,but the cost was still high.Acid precipitation method was rapid with economy and high yield,and fewer impurities.After acid precipitation and methanol extraction,the solution appeared maximum absorption peak in the 199 nm,indicating that the extract of the lipopeptide material.4.The characteristics of antifungal substances of Bacillus amyloliquefaciens TNX-1:Study on the physical and chemical characteristics of antifungal products of TNX-1,thermostable,when the antifungal substances was dealed with 121? for 20 min,it retained some activity;pH-tolerant,the antifungal activity shown no significant difference with the control when dealed with pH2,4,10.When dealed with pH 12 the antifungal activity was in the partial loss of,remained 70.92%of the control;UV-tolerant,after 10 cm at the irradiation from the UV lamp for 30 min the activity of antifungal substances was no significant difference with the control;stable protease,when it was processed with proteinase K and trypsin in a water bath,it remained unchanged in 5 hours.5.Bacteriostatic mechanism of Bacillus amyloliquefaciens TNX-1:When TNX-1 was confront cultureed with Lasiodiplodia theobromae(Pat.)Griff.&Maubl,there was a clear inhibition zone.It would lead the pathogenic hyphae to grow in abnormal shape and produce sac,then it could not further develop into a mycelium,when the concentration of antifungal substances arrived at 300 ?g/mL,it could completely inhibite the germination of pathogen spores.But the antifungal substances had no significant effect on the activity of cellulase and pectinase of pathogens.Therefore,the main mechanism of TNX-1 was the secretion of antifungal substances acted on pathogens directly,which leaded mycelium to deformit in order to control the disease.