Screening And Optimization Of Fermentation Process Of Compound Antagonistic Strain Against Kiwifruit Bacterial Canker

Kiwifruit bacterial canker(KBC)caused by Pseudomonas syringae pv.actinidiae(Psa)seriously constrains to the development of high quality and efficient kiwifruit industry.At present,the control of kiwifruit canker was mainly based on copper preparations and there was a lack of available biocontrol resources.However,the scientific combination of biocontrol bacteria,with multiple biocontrol mechanisms acting in synergy,could reduce the environmental dependence of single biocontrol bacteria strain and more efficiently in disease control.To this end,the group has carried out a lot of research on biological control resources and explored efficient control techniques for kiwifruit canker.Based on the seven Psa antagonists screened in the early stages of the study,the combination of antagonistic bacteria that has a significant inhibitory effect on Psa was screened by in-dish experiments,and its preventive effect on KBC was determined by indoor and field trials.Then its biological control mechanism was initially investigated by biological properties,potted tests and q RT-PCR techniques Subsequently,the biomass of the fermentation broth was used as an indicator to optimize and validate the fermentation process parameters of the strain with the help of single-factor tests and response surface analysis.The main findings of the study were as follows:1.A combination of antagonistic bacteria with good compatibility and strong inhibitory effect,named BD18,was screened from seven strains antagonistic to Psa by compatibility assay and inhibition assay.The combination consisted of Delftia lacustris ZWP15 and Bacillus proteolyticus Z38 at 1:1 with an inhibition diameter of 33.10±0.58mm.The efficacy of BD18 against KBC was 81.72%and 88.97%respectively by vacuum infiltration inoculation of indoor leaf discs and isolated branches wounded inoculation,and it was not pathogenic to kiwifruit.In field trials,the efficacy of BD18 fermentation solution10X against KBC was 73.89%and 81.78%for the main stem,which is significantly better than that of commercially available Bacillus subtilis wettable powder 100X.However,it is not significantly different from the efficacy of 6%vincristine wettable powder 100X and46%copper hydroxide wettable powder 100X.This indicated that the compound antagonist BD18 was effective in preventing KBC.2.By measuring biological characteristics of the two strains,it was found that strain ZWP15 could produce iron carriers and cellulase and it can also decompose organic phosphorus,while Z38 has a strong ability to produce protease and cellulase.Both strains were moderately salt-tolerant and have drought tolerance,biofilm formation and motility.Subsequently,q RT-PCR was used to quantify the effect of BD18 on the expression of genes related to disease resistance in kiwifruit.It was found that Ac ERF1,Ac RCAR1,Ac PAL and Ac RBOH were all differentially up-regulated after inoculation with BD18 and the trend was consistent or significantly up-regulated compared with that inoculated with a single strain.In addition,the results showed that after inoculation with BD18,the plant height of potted tomato(S.lycopersicum)was 27.40±0.82 cm and the root length of potted tobacco(N.benthamianai)was 29.23±3.51 cm,which were 1.70 and 3.25 times higher than those of the blank control.It was clear that BD18 may have multiple disease prevention mechanisms such as competition,lysis,resistance,promotion and induced resistance.3.Through the single-factor test,the most suitable medium for the fermentation of ZWP15 and Z38 was initially screened as TSB medium and LB medium.Through the optimization of medium composition and culture conditions,the most suitable carbon source for the fermentation of ZWP15 was potato starch at 0.5%,nitrogen source was cottonseed cake flour at 1.0%,and the optimum fermentation conditions were in 28℃,at rotational speed of 220 r/min,initial p H value 6.5,inoculum level 3%,and in 18 h.The optimum carbon source for fermentation of strain Z38 was potato starch at 0.5%,nitrogen source was peanut cake flour at 2.0%,and optimum culture conditions were in 32℃,at rotational speed of 220 r/min,initial p H value 7.5,inoculum level 3%,and in 55 h.Through response surface optimization experiments,the optimal fermentation processes of strain ZWP15 were determined as follows:potato starch 0.57%,cottonseed cake flour 0.5%,sodium chloride 0.5%,dipotassium hydrogen phosphate 0.25%,in 24℃,at rotation speed of 205 r/min,initial p H value 7.0,inoculation dosage 2%,and duration 20.2 h.The optimum fermentation parameters of strain Z38 were Potato starch 1.0%,peanut cake flour2.5%,sodium chloride 0.5%,in 28℃,at rotation speed of 234 r/min,initial p H value 7.0,inoculum volume 1.18%and duration 40 h.The dilution coated plate method was used to verify the optimization results of the two strains,and the biomass was 4.23×10~9 cfu/m L and2.90×10~9 cfu/m L respectively.The optimized biomass increased by 91.40%and 84.71%compared with that before optimization respectively.The difference between the actual value and the predicted value was less than 1%,indicating that the established model was stable and effective.The optimized parameters obtained have practical feasibility as well.