| Xenocoumacin 1（Xcn1）is a water-soluble antimicrobial peptide produced by Xenorhabdus nematophila and belongs to the dihydroisocoumarin derivative,which has a strongly alkaline guanido in its structure.Dihydroisocoumarin derivatives represent a series of phenolic antibiotics with extensive biological activities.For the reason of its excellent properties such as strongly alkaline and high stability,guanido compounds have promising prospect in antimicrobial,antiviral and diabetes treatment.Xcn1 has relatively strong anti-microbial activity,especially has remarkable restrictive effect on plant pathogenic fungus and has potent antitumor and anti-ulcer activity effects in medicine.Xcn1 possessed potential used as a new pesticide because its high biological activity and unique structure.The low production of this compound limits its further research and development.Based on this,Xenorhabdus nematophila YL001 which is autonomously isolated and identified was used as a production strain,and systematically studied its shake flask fermentation process.The precursor substances which can increase the yield of Xcn1 were found based on biosynthetic pathway of isocoumarin antibiotic Xcn1.Finally,a novel microbial fungicide with isocoumarin antibiotic Xcn1 as active ingredient was preliminarily developed.The main results of this paper are as follows:（1）The culture broth of the six species of entomopathognic bacteria all showed strong activity on six pathogenic fungi and five pathogenic bacteria,Among which,X.nematophila YL001 exhibits good activity against pathogenic bacteria and fungi.The diameter of antibiotic circle of strain YL001 fermentation broth against Pseudomonas syringae pv actinidiae and Xanthomonas campestris was 29.60 mm and 29.20 mm;the inhibition rate of Botrytis cinerea and Cytospora mandshurica was 100%.The methanol extract of fermentation broth showed excellent protective and therapeutic effect on Botrytis cinerea（Tissue experiment）,the control effect is 65.33%,and the therapeutic effect is56.60%.Compared with other entomopathognic bacteria,X.nematophila YL001 has further research value.（2）X.nematophila YL001 was used as the production strain of Xcn1.High performance liquid chromatography（HPLC）analysis method of Xcn1 was established to determine the difference of Xcn1 produced by different X.nematophila,and the influence of different media on Xcn1 synthesis was analyzed to determine the fermentation base of Xcn1.On the basis of this,the single factor test method was used to investigate the effects of different carbon sources,nitrogen sources and inorganic salts on the production of Xcn1.The fermentation medium used Xcn1 biosynthesis was further confirmed by Central Composite Design（CCD）and response surface method（RSM）.Optimum medium was:20.83 g of Proteose peptone,12.74 g of maltose and 3.77 g of K2HPO4.Under the optimal conditions,the yield of Xcn1 reached a maximum by 113.65μg/mL.Compared with the initial fermentation culture TSB（50.67μg/mL）and basic fermentation culture PP3（87.21μg/mL）,the Xcn1 content was increased by 112.65%and 30.32%;（3）Based on determining the optimal medium,the single factor experiment determined the effects of initial pH,inoculum size,temperature,rotation speed,liquid volume and fermentation period on the yield of Xcn1.The optimal fermentation conditions were initially determined to be initial pH 7.0,inoculum size 10%,temperature 25℃,rotation speed 150 rpm,liquid volume 75 mL/250 mL,and fermentation period 48 h.Under this condition,the yield of Xcn1 reached 153.56μg/mL,which was 35.12%higher than that before optimization.（4）The effects of Xcn1 synthesized precursors（arginine,leucine,urea and acetic acid）on their yield were investigated.The results showed that the four precursors had certain effects on the synthesis of Xcn1,among which Leucine has the most significant increase in Xcn1 production,followed by arginine and acetic acid,and urea is small.The optimum addition concentration of leucine,arginine,urea and acetic acid were 1,3,3 and 2 mmol/L.Under optimum concentration,the yields of Xcn1 were 140.49,132.78,127.30,and 125.70μg/mL,respectively,increased by 42.48%,34.67%,29.11%,27.48%compared with it before adding（98.60μg/mL）.The optimal addition time of the four precursors was 12 h,and the yield was 173.99μg/mL which added precursor at this point,increased by 76.46%compared without precursors,and increased by 243.38%compared with before process optimization.（5）A novel microbial fungicide,1.2%Xcn1 aqueous solution（AS）,was preliminarily developed with antibiotic Xcn1 as active ingredient,through screening different surfactants,antifreezes and preservatives.The formula of agent:1.2%isocoumarin antibiotic Xcn1（active ingredient）、15%OP-10（surfactant）、1%glycol（antifreeze）、1%posassium sorbate（preservative）、91.8%water（solvent）.Indoor pot experiment indicated that1.2%Xcn1 aqueous solution（AS）had a good control effect against rape Sclerotinia disease.In vivo experiment,protective and therapeutic action of 100 times diluent was 54.17%and50.95%.In conclusion,this article improved antibiotic Xcn1 production by the fermentation process of antibiotic Xcn1 by Xenorhabdus nematophila YL001 and the addition of precursor substances.In addition,a microbial fungicide was developed based on this study.These results provide theoretical basis and technical support for industrial production of antibiotic Xcn1. |
