| Longan(Dimocarpus longan Lour.)is one of the main subtropical fruits in China.Since longan fruit matures during the hot season,it is easy to brown,deteriorate and even decay after harvest,which show a serious effect on its edible value and commodity value.However,people are mainly using chemical treatment to prolong its storage and shelf life,there is a need to find alternative non-chemical treatments.In this study,we isolated endophytic bacteriums from longan,litchi,peach,Chinese olive,etc.We have got a strain named LY-1 which antifungal effect better than BS-2.Biology of LY-1 has been studied in this paper.Interactions between LY-1,fungal pathogen and longan fruits also have been studied.The results were as fellows:1.Separation and identification of the antifungal bacteria:We got 126 antifungal bacteria from longan,litchi,peach,Chinese olive,etc.LY-1(One of the antifungal bacteria)was identified as Bacillus amyloliquefaciens base on 16s rDNA sequence analysis,physiological and biochemical data and amplified results with species-special primers(Bsu,Bam and Multiplex).2.Fermentation conditions of LY-1:The single factors including temperature,carbon sources,nitrogen sources and initial pH were studied for producing antifungal target product.We got the optimum fermentation conditions by orthogonal test and verification tests.the optimum fermentation conditions were:Medium:2%of soybean meal,0.5%of ammonium acetate,K2HPO40.1%,KCl 0.050%,MgSO4·7H2O 0.05%,FeSO40.001%and mannitol 0.2%;Temperature:35?;inoculum size:2%;Ventilation:75 mL/150 mL;Speed:170 rpm;Incubation time:72 hours.3.Extraction methods of LY-1 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 206 nm,indicating that the extract of the lipopeptide material.4.The characteristics of antifungal products of LY-1:Antifungal products of LY-1 can endure 121? for 10 min.It still retained 66.6%antifungal activity.There is no significant difference in antifungal activity between treatment and control by deal with pH2.After 20 min irradiated by UV lamp far from 10 cm,the activity of antifungal substances was no significant difference between the control.The antifungal products was processed with proteinase K and trypsin in water bath,it remained unchanged in 5 hours.5.Antifungal mechanism of LY-1:When LY-1 was confronting cultureed with Lasiodiplodia theobromae(Pat.)Griff.&Maubl and Phomopsis longanae Chi.,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 50 ?g/mL,it could completely inhibit the germination of pathogen spores.But the antifungal substances had no significant effect on cellulase and pectinase of pathogens.Therefore,the main mechanism of LY-1 is the antifungal substances acted on pathogens directly,which leaded deformity to mycelium in order to control the disease.6.Physiological and biochemical Effect on longan fruits inoculated by LY-1 and Phomopsis longanae Chi.:(1)LY-1 can reduce the disease index and browning index,as well as respiratory intensity of longan fruit.LY-1 can also delay the senescence of longan fruit by inhibiting the growth of plant pathogens.So it can increase storage time and shelf-life.(2)The antioxidant enzymes(SOD,CAT,APX)activities and antioxidant substances(AsA,GSH,flavonoids)content of the pericarp were increased by the treatment of LY-1,then the ability of the peel to scavenge active oxygen was enhanced and the production rate of O2-.was decreased.The treatment of P.longanae enhanced the ability to scavenge active oxygen but couldn't clear the massive accumulation of O2-,which promoted the lipid peroxidation and rised the MDA and LOX.As LY-1 weaked the poisonous effect on peel from bacteria,the co-treatment delayed the membrane lipid peroxidation and inhibitioned the increasing of LOX activity and MDA content,but made a higher membrane lipid peroxidation of the peel than the treatment of LY-1 individualiy.(3)LY-1 could induce the longan fruit to produce the reaction of stress and enhance the activitie of PPO that promoted the synthese of phenolic substances and made the peel enhence the ability to resistant pathogenic invading.The reaction of stress was the same as its production by pathogenic invading.Because LY-1 could inhibit the growth of the pathogenic,the degree of the structure of cells compartmentalization of the longan fruit was lower,that showed the lower degree of browning on the appearance of the peel.The treatment of P.longanae consumed a lot of phenolic substances of the longan peel and severely damaged the cell structure,as the increasing of PPO activity,the enzymatic browning exacerbatied and the brown of peel increased.The degree of browning of the co-treatment was closed to the control due to that the LY-1 inhibitioned the consumption of phenolic substances casued and destruction of cell structure by bacteria,and the gentle changed of PPO activity,the cell structure integrity was closed to the control too.(4)In the aspects of disease resistance,LY-1 inoculationed for one day all induced the peel to increase the activities of POD,PAL,CHI and GLU,which was the self-defense reaction of the fruit at an aim to accelerate biosynthesis of lignin that can enhance enhanced cell wall's mechanical strength and trengthen the attack of peel to germs,then enhanced the resistance to disease and reduced the disease situation.The treatment of P.longanae increased the activities of disease resistance enzymes and synthesized a large number of Lignin.As the activity of PAL rapidly declined,causing the physiological metabolic of Lignin imbalanced and the lignin degradated to hazardous substances on bacteria,while the activities of CHI and GLU rapidly declined,weakened the attack of peel to germs;except for the first day after inoculating LY-1,the increased of POD,PAL,CHI and GLU activities became slower later,which showed that LY-1 could have a long effect on the pathogen and inhibited the process of corruption of the longan fruit. |
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