Identification Of Elicitors To Induced Plant Systemic Resistance By Bacillus Amyloliquefaciens SQR9

Bacillus amyloliquefaciens SQR9 is a plant growth-promoting rhizobacteria isolated from the rhizosphere soil of a healthy cucumber.and has been used as a biocontrol agent in a commercial bio-organic fertilizer for suppressing cucumber wilt disease caused by Fusarium oxysporum f.sp.cucumerinum J.H.Owen.SQR9 can promote plant growth,enhance plant salt tolerance,and has resistance to several soil-borne diseases.The previous research on the biocontrol function of SQR9 has mainly focused on the competitive colonization and the production of antibiotics,which could directly inhibit phytopathogens.Plant growth-promoting rhizobacterias have been reported to produce various elicitors that induce plant systemic resistance,but there is little knowledge concerning the relative contribution of multiple elicitors from a single beneficial rhizobacterium to induced systemic resistance in plants and the interactions of these elicitors with plant signaling pathways.The present study aims to understand the mechanism of the systemic resistance by SQR9.This study revealed that the colonization of SQR9 triggered innate immunity by accumulating of ROS and malondialdehyde,increasing several defense enzyme activity in cucumber maize and Arabidopsis.For induced systemic resistance,SA,JA and ET are accumulated in roots and shoots of Arabidopsis.Fengycin,bacillomycin D,surfactin,bacillaene,macrolactin,difficidin,bacilysin,IAA,polysaccharides produced by SQR9 induced resistance to inhibited the infection of Pseudomonas syringae pv.tomato DC3000 and cucumber Botrytis cinerea on plant leaves.Among those eliciltors,surfactin,macrolactin and bacilysinplayed the major roles in ISR,those elicitors showed specific effects on plant defense pathways against different phytopathongens.The colonization ability of SQR9 onArabidopsis root and maize root were not the same with that on cucumber root.The population of SQR9 in cucumber maize and Arabidopsis root surface were 5.70×105CFU/cm2,5.62×102CFU/cm2and 2.26×103CFU/cm2 respectively.After 6 or 12 hours post inoculation,O2·-and H2O2,respectively,accumulated in leaves of these plants.Then,the expression of AsA-GSH cycle genes wereactivated,and scavengation of reactive oxygen species was initiated to relieve oxidative damage in cells.At the same time after inoculation of SQR9,the content of malondialdehyde,a marker material of membrane lipid peroxidation was increased,plants were suffered with stress.SQR9 inoculation significantly increased the activities of defense enzymes including ?-1,3-glucanase,lipoxygenase,peroxidase in Arabidopsis,and?-1,3-glucanase,lipoxygenase,polyphenoloxidase incucumber and maize.Those changes showed that the plant defense system was activated.Inoculation with SQR9 led to a significant decrease of population of Pst DC3000 and Botrytis cinerea by inducing plant systemic resistance.It was observed that root content of SA,JA and ET increased to the highest(1.4-fold,2.0-fold and 1.4-fold of those in CK)at 1 day after inoculation with SQR9,after which the content of SA and JA decreased;however,ET content in SQR9-inoculated root was still significantly higher than that in the CK root after 4 days.It should be noted that shoots showed a slower response than roots did.The content of SA and JA in shoots gradually increased until reaching 1.3-fold higher than the CK at 4 days,while ET increased to 1.2-fold at 2 days.The transcription of ISR-related genes increased to the highest at 6 to12 hours post inoculation.Mutation of the Arabidiopsis genes involved in SA,JA and ET signaling transduction decreased the resistance induced by SQR9.The results indicated that all three signaling pathways in Arabidopsis were activated by inoculation of SQR9.SQR9can produce multiple antibiotics and secondary metabolites,include lipopeptide(bacillomycin D,fengycin,surfactin),polyketide(bacillaene,macrolactin,difficidin),dipeptide bacilysin,2,3-butanediol,IAA and exopolysaccharide.SQR9 mutant deficient in the production of each potential elicitor was tested for their function in inducing systemic resistance.The results showed that mutation of the production of lipopeptide and polyketide(?sfp)caused a sharp decrease in the ability of SQR9 to induce plant resistance against Pst DC3000 and Botrytis cinerea.For single antibiotics,mutants deficient in surfactin,bacillomycin D,fengycin and macrolactin production showed half the ability of SQR9 to induce plant resistance against Pst DC3000,and mutants deficient in bacillomycin D,surfactin,difficidin,bacillaen,IAA,exopolysaccharide and bacilysin showed significantly higher contributions to plant resistance against Botrytis cinerea than other antibiotic.These secondary metabolites could act as elicitors to induce plant systemic resistance,moreover,these elicitors had specific effects on the induction of plant defense pathways against different phytopathogens.Moreover,the correlation of bacterial genes and plant defense genes and the crosstalk between hormones and signaling pathways was studied.Surfactin?macrolactin and bacilysin had strong ability to trigger ISRin Arabidopsis.After treated with purified elicltors,plant showed different resistant against Pst DC3000 and Botrytis cinerea.Surfactin and macrolactin played important roles in protecting plants from Pst DC3000 and Botrytis cinere,whilebacilysin showed a weaker effect.Transcriptome of Arabidopsis treated with pure surfactin,macrolactin and bacilysin showed that surfactin and macrolactin caused decrease of genes involved in photosynthesis,and increase of genes involved in glucose metabolism and chitin degradation in plant.All of those responses were suitable for plant to active resistant to pathogens.Nevertheless,macrolactin and surfactin showed differences in activation of phytohormones signaling pathways,include auxin,cytokinin,gibberellin,abscisic acid,brassinosteroid,ethylene,jasmonic acid and salicylic acid.In addition,shoots responses are stronger than roots.This study revealed that SQR9 could trigger innate immunity and induce systemic resistance againstPst DC3000 and Botrytis cinerea.SA-,JA-and ET-signaling pathways were involved in the ISR response activated by SQR9.SQR9produced different elicitors to induce different kind of defense pathway against pathogens.