Functions And Mechanisms Of The New Lysobacter Enzymogenes Strain LE16 In Plant Growth Promoting And Disease Biocontrol

China consumes 2.5-2.6 folds of chemical fertilizers per unit area on average compared with United States and the European Union and 2.5 times of chemical pesticides compared with global average.In contrast,their efficiencies are much lower than the mean values worldwide.The repeatable and chronic use of these chemicals have caused serious problems related to crop cultivation,environment quality and human safety,including soil productivity deterioration,heavy metal accumulation in arable lands?mainly derived from chemical phosphate fertilizers?,water eutrophication,decreased biodiversity,pathogen's drug resistance?or invalidation?,and food contamination.Under the conditions of continuous cropping for high crop yield,sole dependence on traditional techniques is far enough to reduce the application of chemical fertilizers and pesticides.This necessitates the innovative ideas,new technologies and products.Microbial biotechnologies are powerful ways to mobilize soil nutrients,promote plant growth,enhance crop resistance against diseases,and improve efficacies of fertilizers and pesticides.They can greatly reduce the use of chemical fertilizers and pesticides.Microbial products are human-safe,resources renewable,and environment-friendly but are less in categories,poor in efficiencies,and expensive in costs.It is necessary to search for more effective microbes,reduce the production costs,and increase the efficacy of microbial inoculants.Therefore,a novel bacterium with multiple functions was initially isolated from a field with long-term flue-cured tobacco,canola,and rice rotation in Yunnan Province,China,identified as Lysobacter enzymogenes based on its physiological and biochemical characteristics and whole genome sequence and designated as strain LE16.Then the effects of L.enzymogenes LE16 on plant growth and disease biocontrol as well as the related mechanisms were investigated by the ways of biomolecular,chemistry,plant pathology,pedology and plant nutrition.The results obtained would provide us some scientific basis and a new way for the development of sustainable agriculture.Following are the main results obtained.?1?Taking into account the production of phosphatase,protease,lysozyme,indole-3-acetic acid?IAA?,and siderophore,it is reasonable to predict that L.enzymogenes LE16 might mobilize soil organic phosphorus?P?and nitrogen?N?,whereby increase availability of P and N in soils,and antagonize phytopathogens,by which control plant diseases.Autolysis occurred after the bacterial isolates were grown in liquid medium for several days,which has never been observed with known Lysobacter species.Furthermore,16S rRNA gene and whole genome sequencing indicated that L.enzymogenes LE16 is a new strain and different from the identified L.enzymogenes OH11,L.enzymogenes C3,and L.enzymogenes 3.1 T8.Bioinformatics found serval functional and regulatory genes related to the production of lytic enzymes,siderophore,IAA,and antimicrobial compounds existed in the nuclear genome of L.enzymogenes LE16,showing the potential of plant growth promotion and disease control.?2?L.enzymogenes LE16 was able to simultaneously produce acid,neutral,and alkaline proteases in liquid medium and hydrolyze bovine serum albumin?BSA?to NH₄⁺.The BSA hydrolysis capacity increased with incubation temperature at 12°C-28°C,but varied very little in the solutions at pH 4.0-10.0.In addition,the bacterial isolates were capable of producing extracellular acid,neutral,and alkaline phosphatases in the liquid Mongina medium,through which effectively removed P from lecithin in different solution pH?4.0-10.0?and incubation temperature?12°C-28°C?.The hydrolysis reaction was almost unaffected by the addition of NH₄⁺,NO₃^-,and urea into the culture solution at equivalent N concentrations although proper amounts of exogenous N and inorganic P significantly increased the hydroxylation efficacy.?3?L.enzymogenes LE16 successfully survived and effectively mobilized organic N and P in a purple soil via the production of phosphatases and proteases.After incubation for 30 days,1N NaOH-hydrolyzable N,NH₄⁺,Olsen P,and water-soluble P in the inoculated soil were increased by 17.82%-22.26%,46.54%-47.86%,64.33%-81.67%,and 48.82%-55.88%,respectively,compared with the soil without inoculation.?4?L.enzymogenes LE16 inoculation significantly increased plant root activities as well as nutrient uptake and promoted growth of lettuce and pepper seedlings in greenhouse pot experiments.Compared with only chemical fertilizers,application of bacterial inoculant in combination with chemical fertilizers increased the yields of lettuce and pepper by6.43%-11.30%and 43.82%-70.33%,respectively,in addition to quality improvement.The chief mechanism employed by this bacterium could be the mobilization of organic N and P,by which increased the availability of these two nutrients in the test soil.?5?When L.enzymogenes LE16 was grown in the liquid medium at the same size of 50mL,the time of bacterial complete autolysis was reduced from 336 h at 12?to 96 h at 36?.However,it was decreased from 216 h in 400 mL culture solution to 120 h in 50 mL culture solution at the same incubation temperature of 28?.Culture solution pH?5.0,7.0,and 9.0?and inoculation proportion?1%,2%,and 5%?had no effect on autolysis occurrence.Therefore,the mechanism of L.enzymogenes LE16 involved in autolysis may be quorum sensing,which randomly occurred in a single cell and then spread through cell-cell contact,finally resulting in the disappearance of all microbial cells.?6?L.enzymogenes LE16 inhibited successfully the growth of Staphylococcus aureus,Pseudomonas syringae pv.tabaci,Colletotrichum gloeosporioides,Penicillium italicum,Alternaria alternate,Rhizoctonia solani,Didymella bryoniae,Sclerotinia sclerotiorum,Phytophthora nicotiana,and Phytophthora capsici.H-SDS?heated SDS at 100?for 30 min?and S-SDS?stored SDS at room temperature for 12 months?also showed strong antagonistic activities against most plant pathogenic fungi and oomycetes and caused mycelia abnormality,and their antifungal activities may be attributed to the production of protease,phosphatase,lysozyme,siderophore,and antimicrobial compounds with thermostability by L.enzymogenes LE16.?7?The application of SDS induced plant gained systemic acquired resistance and reduced flue-cured tobacco blank shank and pepper blight disease incidences by 54.96%-75.67%and86.20%-93.10%,respectively.Foliar spray of SDS significantly decreased the infection of cucumber leaves by D.bryonia and achieved control efficacies of 50.65%-53.67%,which were similar with chemical pesticide thiophanate-methyl.SDS,H-SDS,and S-SDS strongly inhibited the conidial germination of Erysiphe cichoracearum and Sphaerotheca fuliginea and successfully control powdery mildews of flue-cured tobacco and cucumber,with control efficacies of 92.25%-100%and 91.30%-96.78%,respectively,in greenhouse pot experiment.SDS also showed a satisfied and sustained control effect against flue-cured tobacco powdery mildew in the field.In general,L.enzymogenes LE16 was able to mobilize soil organic N and P,promote plant growth,antagonize variosus pathogenic microrgannisms,and control plant diseases and powdery mildews,which is promising in practical use.