Isolation And Identification Of The Major Bioactive Compounds Of Bacillus Atrophaeus HAB-5 And Mechanism Of The Antimicrobial Activity

Beneficial microorganisms such rhizobacteria are well known for their inhibitory effect against different phytopathogens.The useful biological activities of their produced secondary metabolites have received considerable attention.Among these microorganisms,Bacillus species are recognized for production of different compounds and displayed interesting antagonistic effect.The bacterial strain designed as HAB-5 was isolated from soil of cotton field,Xinjiang,China and has remarkably exhibited a broad spectrum of antimicrobial activity.The present study is an effort to elucidate the concept of the isolate HAB-5 in the current scenario and underline the possible mechanisms involved for its antimicrobial activity.We have identified the isolate.The main compounds responsible for its activity was purified,identified and characterized.Moreover,we have optimized the compounds production.We also have evaluated the effectiveness of HAB-5 for promoting plant growth and controlling crop disease.Finally,to ensure its application,eco-toxicological and safety evaluation was assessed in zebra fish.The obtained result are summarized bellow:1.Through morphological,biochemical,physiological characterization and molecular characterization included 16S rRNA gene sequence analysis and specific primers cloning,the isolate HAB-5 was identified as Bacillus atrophaeus.2.The ability of the strain HAB-5 to promote plant growth and to control disease was evaluated.The result showed that HAB-5 was competent of expressing multiple PGP(Plant Growth Promoting)traits.It had a great potential for production of 1-aminocyclopropane-l-carboxylic acid(ACC)deaminase.The strain HAB-5 grew well in the medium containing ACC while it did not grown the medium without ACC.On Pikovskaya's agar medium,the development of a clear zone encircling the bacterial growth indicated phosphate solubilization.Based on FeCl3 test,appearance of orange brown color produced by the cell filtrate indicated the siderophore production of the isolate.Proteolytic activity was tested in LB agar plates supplemented with 3%skim milk powder,and the apparition of clear zone surrounding the bacterial suggested protease production.For cellulase production,HAB-5 was grown on carboxymethyl cellulose(CMC)agar medium and then stained with 0.1%Congo red.After staining,agar plates were observed for zones around the colonies.These results indicated that HAB-5 was candidate for grown promotion and had good activities on plants pathogens.3.The isolate was assessed for antifungal activity using different protocol.The result showed that HAB-5 had broad spectrum antifungal activity against 25 plant pathogenic fungi.The best activity was recorded against Colletotrichums gloeosporioides Penz MgCg-1 by 67.23%,followed by Fusarium proliferatum,Alternaria alternata and Botrytis cineria 62.87%,61.73%and 55.10%respectively.Second,the agar plug containing the HAB-5 released metabolites harvested around the bacterial growth showed good inhibition of C.gloeosporioides mycelia growth by 37.36%and 39.31%in dual culture and single culture respectively.Third,HAB-5 also capable to produce the volatile organic compounds(VOCs)that can inhibit the growth of different pathogenic fungi by 74.41%,70.79%,51.30%,50.13%,43.57%in Alternaria alternata and Fusarium oxysporum,Phytophtora nicotinae,Colletotrichums gloeosporioide and Fusarium graminearum respectively.Furthermore,it was able to export the bioactive substances from the cytoplasm to the extra-cellular environment.Cell filtrate from different culture medium were found have strong antifungal activity.4.The more effective bioactive antifungal metabolites of HAB-5 was harvested from the petroleum ether extract using liquid-liquid extraction protocol.The crude extract had a potential activity against C.gloeosporioides.In vitro experiment,the inhibition zone diameter was reached to 27.13± 1.25 mm.Also,it induced morphological abnormalities in hyphae of C.gloeosporioides.5.For purification of the bioactive compounds,the crude extract metabolites(16g)was subjected to the different separation using column chromatography.Two major bioactive fractions were obtained.The fraction I was subjected to Gaz Chromatography/Mass spectrometry(GC-MS)analysis.We have identified 19 volatile organic compounds included one esters,two alcohols,two alkenes,height alkanes,and six organic acids.Interestingly,we found that these can work individually or with cooperation to accomplish their antifungal activity.Suggesting that there is synergistic effects occur between these compounds to accomplish the disease control.In contrast some compounds was found had incompatible association.Their mixture abolished their inhibition effect.Moreover,B.atrophaeus HAB-5 produced compound that had completely antagonize all test organisms.This finding bring a little for understanding the mechanism of the antagonistic organism to control the pathogens growth.6.The partial purified fraction II was analyzed in Liquid Chromatography/Mass spectrometry(LC-MS).The result revealed identification of the lipopeptides including iturin and mycosubtilin.The genomic DNA of HAB-5 was subjected to screening for the presence of the lipopeptide biosynthetic genes.Six genes included BAC,ituC,FenB,mycB,itu A and lpa have cloned.7.The antifungal compounds were fully heat stable.There was sharp increase in activity with increased of temperature.Antagonistic activity was better by autoclaving at 120? for 20 min.The pH test revealed that compounds exhibit activity in a broad pH range between 5.0 and 10.0 with an optimum of 7.0.However,this activity was found reduced significantly under pH 5.0 and above 10.0.It was also found that after different proteolytic enzymes treatment,the antifungal metabolites did not lose the antifungal effect.MIC(minimum inhibitory concentration)determination revealed that the highest effect was found 34 ?g/mL,42 ?g/mL,55?g/mL in C.gloeosporioides,P.nicotinae and A.solani respectively.Against Botrytis cinerea and F.oxysporum the MIC were 67 and 76 ?g/mL respectively.The effect was reduced in Fusarium gramirearum and Phyllosticta theaefolia in which the value were 201 and 229 ?g/mL respectively.Half maximal effect concentration(IC50)was 148.57 ?g/mL.These results supported the strong antifungal activity of HAB-5.8.Optimization of the antifungal production of B.atrophaeus HAB-5 was investigated using different fermentation conditions.The obtained results were as follow.Luria Bertani and Mueller Hinton culture medium gave the highest yield.Incubation period was 48 hours.Temperature and pH were 28? and between 6.50 to 7.0 respectively.The inoculation quantity was 2%.Incubation speed was 180 rpm.This investigation suggested that the level of antifungal production may be strongly influenced by the type and the nature of the carbon(glucose)and the nitrogen source supplied in the culture medium.9.The capacity of the strain HAB-5 to promote plant growth was evaluated in different condition.The result showed that the most effective effect was found in cell suspension.In vitro experiment,tomato seed bacterized of 1×107 cfu/mL of cell suspensions was enhanced the seed germination by 48%and developed a robust root system with proliferation of lateral roots comparing to control group.Same concentration increased Nicotiana benthamiana root length by 54.71%.It was also observed that the VOCs of HAB-5 significantly increased fresh biomass of tobacco plants by 4.01 fold comparing to the non-inoculated control.In pot experiment condition,HAB-5 was successfully colonized Nicotiana tabacum root.Up to 60 days,1×107 cfu/mL of cell suspension drenched in the root exhibited significant increase in fresh shoot weight,dry shoot weight,fresh root weight and dry root weight,76.47%,80.58%,71.71%,82.10%respectively as compared to the non-inoculated control plant.The inoculated plants were distinctly taller than the non-inoculated plants by 45.76%and displayed much darker green leaves.The role of HAB-5 in plant growth was also reconfirmed by analysis of expansins genes NtEXP1,NtEXP2 and NtEXP6 in the tobacco leaves.HAB-5 up regulated these growth related genes leading to growth promotion.10.1×107 cfu/mL of HAB-5 cell suspension supported the growth of N.tabacum protected the plant from the abusive effect of Tobacco Mosaic Virus(TMV)and also reduced the pathogenecity of P.nicotianae.In tobacco plant at 30 days post inoculation,HAB-5 reduced the P.nicotianae severity kept proline,hydrogen peroxide,nitric oxide,chlorophyll content at higher levels,and keeping the MDA(malondialdehyde)content at a lower level.HAB-5 treatment also up regulated the defense and growth promotion related genes included NtEXP2,NPR1,Hin1,then at the end leading induced systemic resistance.11.The capacity of the crude extract form HAB-5 to control plant disease was also investigated.It was found that 1 mg/mL of the crude extract metabolites released by HAB-5 was effective to lowered the severity of anthracnose on mango fruits and protect tobacco seedling from P.nicotianae.It also has reduced the virulence of TMV in tobacco leaves.The signaling regulatory gene(NPR1),defense genes(PR-la,PR-1b,Chia5),and hypertensive response related genes(1Hsr203J,Hinl)were up-regulated in plants treated with the metabolites.Furthermore,the VOCs of HAB-5 was significantly reduced the pathogenecity gray mold on tomato fruits.Altogether,these accumulated results strongly supported the strain HAB-5 to be a biocontrol strain.12.The present research work was undertaken to investigate the response of zebrafish embryos to the biological pesticide extracted from the strain HAB-5 after exposition to the different concentration(49,20,61.40,72,80,96.00 mg/L and 120 mg/mL)from 24 hours post-fertilization(hpf)to 96 hpf.The result showed that LCso value of metabolites for D.Rerio embryos was 103.92 mg/L,with 95%confidence intervals range 89.79-130.58 mg/L.Except in high exposed concentration at latest time point,all exposed embryos were fully developed within 72 hours without deformities.The metabolites was not toxic on exposed embryos.However the highest exposure concentration(120 mg/mL)at 96 hpf caused slight developmental abnormalities such decrease in movements and in heart beats,small effect on final hatchability.In contrast,it did not display effects on mortality.The overall results indicated insights into the potential safety of the strain HAB-5 for crop protection.