Screening And Identification Of Endophytic Antagonistic Bacteria From Sweet/Brown Rot Of Sweet Cherry And Study On Antibacterial Active Substances

Prunus avium L.is rich in nutrition and bright in color.It is one of the most popular temperate fruits among consumers.However,during the post-harvest storage process,dehydration,softening,browning and pathogens are prone to occur due to the thin skin and the juice,which would lead to fruit rot.Among these factors,soft rot and brown rot are main diseases of sweet cherry after harvest.Therefore,it is of great research significance to isolate and identify the pathogens of the two diseases.In this test,"Mei Zao"sweet cherries with a high market share in Shandong were taken as the research object."Mei Zao"sweet cherries with soft/brown rot characteristics were collected to isolate pathogens which were collected from Tai’an,Shandong province.The morphology and molecular biology were used to identify the pathogenic strains of sweet cherry and pass them back to the sweet cherry for pathogenicity verification.For soft/brown rot pathogens,select high-quality antagonistic strains from the healthy sweet cherry itself and carry out biocontrol research.The following results are obtained:(1)Twenty-one strains of fungi were isolated from 60 rotten"Mai Zao"sweet cherry samples which were collected from Tai’an,Shandong province,and the spoilage characteristics of sweet cherry caused by RF and HF pathogenic fungi were consistent with the onset of sweet cherry soft/brown rot.The reverse connection verification test showed that the pathogenicity of colony RF was stronger than that of colony HF.The RF strain was identified as Rhizopus stolonifer,and the HF strain was Monilinia fructicola.The study of biological characteristics showed that Rhizopus solani had the best colony growth and spore production under SDA medium with yeast extract as nitrogen source,at the temperature of 25℃,p H=6 and dark conditions.(2)After morphological identification,physiological and biochemical indexes,and 16S r DNA sequence analysis,the endogenous antagonistic bacteria L-16 and L-21 isolated from the interior of sweet cherry were identified as Bacillus velezensis and Bacillus tequilensis.In the optimization of the bacteriostatic conditions of the antagonistic antibacterial L-16 fermentation broth,it was found that under the fermentation temperature of 37℃,p H of 6.5,and time of 96h,the effect of RF on pathogenic bacteria was the best.The L-16 of antagonistic bacteria was tested in vivo to verify the biocontrol effect on the RF of pathogenic bacteria.The results showed that the antagonistic effect of the fermentation broth was better than that of the bacterial suspension and the fermentation supernatant.The rot rate of sweet cherry was 20.58%,the diameter of the lesion was 6.77±1.41 mm,and the disease index was 13.97%afte 48 hrs’inoculating the fermentation broth at a concentration of 1×10~9 CFU/m L,which were significantly different from the control group(p<0.05).(3)Interaction of antagonistic antibiotic L-16 and pathogen RF:Scanning electron microscopy(SEM)was used to determine the effect of antagonistic antibacterial treatment on bacterial mycelium rupture and deformity and depression of sporangia.SEM in vivo experiments showed that antagonistic bacteria would multiply in the wound site,and closely adhere to the pathogenic hypha,which may due to the competition of nutrition and space.(4)The research on the stability of antibacterial substance L-16 against temperature,p H,ultraviolet and enzymes illustrated that the antibacterial substance of strain L-16 had the highest antibacterial activity at p H=7.Under different ultraviolet irradiation time,the antibacterial activity basically did not change significantly,which showed a good ultraviolet stability;Compared with different temperatures,the bacteriostatic effect was in the best condition at room temperature,in addition,with the increase of temperature,the bacteriostatic activity would gradually decrease.The biological activity of the antibacterial substance would be completely eliminated at a temperature of 60℃.Under different enzyme treatments,compared with CK,the activity of L-16 antagonistic antibacterial substances was significantly different.It can be preliminarily determined that the antibacterial substance produced by L-16antagonistic bacteria may be proteins or lipopeptides.Salting out the antagonistic bacteriostatic substance,it was found that when the ammonium sulfate saturation was 80%,the supernatant had no bacteriostatic activity,and the precipitated substance had the best bacteriostatic ability.