| Apple Valsa canker(AVC),caused by the weakly parasitic ascomycete,Valsa mali Mayabe et Yamada is a long-standing concern during apple production.All commercial apple cultivars are susceptible to the disease.The control of AVC is particularly challenging using chemical treatments since the pathogen penetrates deep into the host phloem and xylem,where it is inaccessible to conventional fungicide treatments.Traditional control measures such as canker surgery and pruning infected limbs are also inadequate as these inflict further damage to the trees.Therefore,a new,safer,and more effective method to control of AVC is required.One alternative that has shown promise is biological control,as an effective,environmentally friendly control strategy.Among the currently used biocontrol agents(BCAs),Bacillus species are often regarded as ideal candidates for commercial use since they are effective against pathogens via multiple mechanisms including secretion of various antifungal substances.Additionally,these agents are not easily affected by external environmental conditions due to their ability to form heat-and desiccation-resistant endospores.We previously showed found that Bacillus EDR2 and EM7 exert a strong antagonistic effect against V.mali.In this study,we identified key genes and proteins from EDR2 and EM7 that are related to antifungal activity.The results of this study,as described below,will provide a theoretical basis for further development of these BCA strains for the control of AVC.1.Identification of the antagonistic strain EDR2,characterization of the antifungal substances produced and compared with strain EM7: The strain EDR2 was identified as Bacillus amyloliquefaciens using morphological,physiological,and biochemical characteristics,followed by gene sequencing of three housekeeping genes 16 S r RNA,gyr B and rpo B.Next,proteins were precipitated from the EDR2 fermentation broth by TCA precipitation.The results showed no statistically significant difference in antagonistic activity between the fermentation broth and culture filtrate(P < 0.05),while the supernatant without proteins showed no antagonistic activity.Thus,it was confirmed that antifungal proteins were the main antagonistic substances produced by EDR2.The results of inhibitory spectrummeasurement showed that both,EDR2 and EM7 inhibited mycelial growth of all fungi studied;however,the two antifungal strains showed a statistically significant difference in inhibition effect(P < 0.05)for the same target fungus.Resistance tests showed that both,EDR2 and EM7 showed growth within a p H range of 5 to 10 as well as in the presence of stress conditions such as 1% to 9% Na Cl,but there are significant differences in colony morphology.2.Development and screening of EDR2 and EM7 mutants with significantly altered antifungal activity using genetic transformation techniques: EDR2 and EM7 mutant libraries were constructed by transposon insertion technology,yielding 5256 EDR2 mutants and 413EM7 mutants.Further,the antifungal activity of mutants against Valsa virulent strain WT03-8 was tested by plate confrontation method.A total of 21 mutants with reduced antagonistic activity compared with EDR2 were selected from the EDR2 mutant library,accounting for 0.4% of the total mutants.One mutant,EM-217,that had reduced antagonistic activity compared with EDR2 was selected from the EM7 mutant library and accounted for0.24% of the total mutants.But there were no any mutants that had inhanced antagonistic activity.Polymerase chain reaction(PCR)confirmed that the 22 mutants selected were produced by random insertion of the Tn YLB transposon into the wild-type EDR2 or EM7 genome.Southern blot analysis showed that 20 of these mutants,excluding mutant strains ED-1806 and ED-1818,were single-copy insertions,accounting for 91%.3.Identification of genes from EDR2 and EM7 that were related to antifungal activity:The gene sequence for 18 transposon insertion mutants was obtained using Site Finding-PCR amplification,yielding 8 genes related to antifungal activity,where the functions of 3 were known and 5 were unknown.Of these 8 genes,3 genes with known functions(thioesterase II-like protein,endo-?-1,3-1,4-glucanase,and putative chitin-binding protein)were directly associated with antifungal activity.On the other hand,6(putative oxidoreductase;3-oxo-acyl-ACP reductase [Dlt E];putative nucleoside 2-deoxyribosyl transferase;histone arginine demethylase [JMJD6],?-ketoacyl synthase,and mannose-6-phosphate isomerase)affected the growth or cell membrane structure of strain EDR2 and indirected with antifungal activity.Based on the results from gene knockout and antifungal activity analyses,the unknown functions of 5 genes were found to be closely related to antifungal activity.Thus,these 5 genes were identified as potential new antifungal activity-related genes.4.Expression of 8 genes related to antifungal activity in a prokaryotic system and identification of antifungal proteins: Eight prokaryotic expression vectors were constructed using p ET32a(+)vector.Recombinant proteins were induced using 1 m M isopropyl?-D-1-thiogalactopyranoside(IPTG)at 30°C and sodium dodecyl sulfate-polyacrylamide gelelectrophoresis(SDS-PAGE)results showed that 3 genes(i.e.,those encoding hypothetical protein(from mutant ED-2500),putative chitin-binding protein,and endo-?-1,3-1,4-glucanase)were successfully expressed in Escherichia coli BL21.Recombinant colonies were broken by sonication and proteins in the supernatant and precipitate were detected.Only 1 recombinant protein,Glu-His,encoded by the ?-1,3-1,4-glucanase gene was detected in the supernatant,while the other 2 recombinant proteins encoded by hypothetical protein and the putative chitin-binding protein gene were detected in the precipitate.Further,the antifungal activity and stability of the recombinant protein Glu-His were tested.The results showed that recombinant antifungal protein,Glu-His was heat-and acid-resistant,with properties similar to those of crude proteins extracted from EM7 broth.Therefore,the ?-1,3-1,4-glucanase gene is an important gene related to antagonistic activity and its expression product is an important antifungal substance obtained from EM7 broth.5.Analysis of different extracellular proteins produced by EDR2 and 5 mutant strains:Extracellular proteins from 5 mutants and EDR2 were extracted using trichloroacetic acid-acetone(TCA)method.Different proteins were detected by 2-dimensional electrophoresis and analyzed by PDQuest 8.0 2 Analysis software.A total of 17 proteins were detected in EDR2 only,while 20 proteins showed downregulated expression in the mutants.Next,33 differentially expressed proteins were obtained by mass spectrometry,of which 2proteins were successfully expressed in a prokaryote,purified,and tested for antifungal activity.The recombinant protein AP-His showed strong antifungal activity against V.mali,determined to be a very important antifungal protein.The recombinant protein,UP-His showed weak antifungal activity against V.mali,but the antifungal activity of UP-His knockdown mutants was severely reduced.Therefore,we speculate that UP-His may exert antifungal activity via indirect regulation of other genes. |
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