Study On Optimization Of Fermentation Medium And Components Of The Antibacterial Substance Produced By Pichia Anomala0732-1

Bacteria fruit blotch (BFB), caused by Acidovorax avenae subsp. Citrulli (Aac), has been responsiblefor significant economic losses to cucurbit production and recognized as a devastating disease on Hamimelon as well as as worldwide quarantine disease. Antibacterial substance (ABS) has performed stronginhibition against Aac produced by P. anomala0732-1in our research.Objectives: In order to obtain optimal fermentation system for producing ABS and demonstrate segmentalmode of action of P. anomala0732-1,the work was done from4aspects,optimizing the fermentationmedium, analyzing the components of ABS, measurement of resistance-related enzymes induced by ABSand effect of nutritional compound in biocontrol activity of P. anomala0732-1.Methods: Single-factor and response surface methodology (RSM) were applied to optimizingfermentation medium. ABS were identified by nuclear magnetic resonance spectral (NMR), gaschromatograph-mass spectrometer (GC-MS) and high performance liquid chromatography. Time dynamicof phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), superoxide dismutase(SOD) andperoxidase (POD) were detected after respective inoculating Aac and fermentation liquid of P. anomola0732-1on hami melon. The synergistic effect of this antagonistic yeast in combination with calciumchloride (CaCl2),2-Deoxy-D-Glucose (2DOG) and methyl jasmonate (MeJA) were tested in this workby measurement of inhibition zone and lesion diameter.Results: The optimal fermentation medium estimated for components related to the maximum of inhibitionzone (13.86mm) were41.2g/L sugar,1.39g/L yeast extract,5g/L peptone,1.59g/L MgSO4and1.25g/LKH2PO4。Acetic acid (1.1±0.1%), succinic acid (0.043±0.003%), citric acid (0.033±0.003%), malicacid (0.033±0.003%), lactic acid (0.031±0.004%) and propionic acid (0.011±0.001%) were detected inABS, there is a large amount of acetic acid.Results in vitro showed that CaCl2(2.5%w/v),2DOG (36mM) and MeJA (200μM) significantly improved the capacity of the antagonist to control Aac, CaCl2andMeJA had no any enhancement on the growth of P. anomala0732-1.There was an adverse effect for2-DOG on the population of antagonist P. anomala0732-1.These four enzymes have increasedsignificantly and steadied high level with treatment of P. anomoala0732-1in156h,POD was expressedhighest level, the maximum value of POD reached at432.86U/g approximately1.8-fold that of the control.PAL24h (activity10.72U/g, approximately1.2-fold that of CK), PPO36h (activity4529.20U/g)Conclusions: Inhibiton zone was increased to45.29%by optimal fermentation medium compared withinitial medium. Resistance-related enzymes of PAL、PPO、POD and SOD were highly and robust expressedby incolated with P. anomala0732-1on hami melon seedlings compared with CK, this work support theview that increased resistance-related enzymes could be involved in the mechnism against bacterial fruitblotch by P. anomala0732-1.The addition of nutritional compounds also play a key role in enhancementof biocontrol efficacy of of P. anomala0732-1. The incidence of BFB was markedly reduced by over simplified seed treatments of P. anomala0732-1.These demonstrated that P. anomala0732-1has greatpotencial in biocontrol of BFB.