| So far,spore-forming Bacilli include 729 species,which cover Alicyclobacillaceae,Bacillaceae,Paenibacillaceae,Pasteuriaceae,Planococcaceae,Sporolactobacillaceae and Staphylococcaceae.Up to 47 of species in spore-forming Bacillaceae,Paenibacillaceae and Pasteuriaceae family of Bacillales order were found to have activity of killing nematodes in our knowledge by surveying from literatures.In this study,we identified nematicidal factors and mechanisms and studied the nematicidal trains of 120 nematicidal spore-forming Bacilli which cover 30 species across Bacillaceae and Paenibacillaceae family at genome and metabolome level.Spore-forming Bacilli can affect nematode development,fecundity and survival,for example,killing nematodes.However,although many Bacillus species can kill nematodes,the virulence mechanisms spore-forming Bacilli utilize remain unknown.In this study,we collected 120 strains comprising 30 species across the Bacillaceae and Paenibacillaceae families of the Bacillales order and measured their nematicidal activities.Comparison of these strains’ nematicidal capacities revealed that nine species,including Bacillus thuringiensis,B.cereus,B.subtilis,B.pumilus,B.firmus,B.toyonensis,Lysinibacillus sphaericus,Brevibacillus laterosporus and B.brevis,were highly nematicidal species,the first of which showed the highest activity.Strains of ten species were newly found with nematicidal activity,including.toyonensis,B.stratosphericus,B.muralis,B.galactosidilyticus,Fictibacillus phosphorivorans,F.arsenicus,Paenibacillus larvae,P.alvei,P.dendritiformis and B.agri.We sequenced 115 of these strains through Illumina HiSeq2000 or HiSeq2500(five genome sequences can be acquired from GenBank).The average coverage of the sequenced genomes is 138.8,and the number of scaffolds over 500 bp after assemblying and gap filling is between 54 and 627,the length of scaffolds changes from 19,349 bp of B.thuringiensis to 2,935,418 bp of B.megaterium,the GC content of genomes changes from 34.8%of B.thuringiensis to 54.53%of P.dendritiformis,and the genome sizes change from 3,586,955 bp ofB.stratosphericus to 6,907,172 bp of B.thuringiensis.Genome sequencing and analysis identified plentiful nematicidal factors,which grouped into five types,including crystal proteins(Cry),compounds,proteases,chitinases and other proteins.The distribution of these nematicidal factors show species sepecificity,Cry and thuringiensin exist in B.thuringiensis strains with high nematicidal activity,metalloproteinase Bmp 1,collagenase ColB and chitinases are mainly in B.cereus(Bc)group,protease Bae16 exists in Bc group,B.megaterium,B.aryabhattai,B.muralis,P.larvae and B.agri,protease Bace16 exists in B.licheniformis,B.atrophaeus,B.stratosphericus and B.endophyticus,but Bacel6 and Bae16 coexist in B.subtilis(Bs)group.At least four mechanisms were deduced on the basis of combination of these factors and the bacterial nematicidal activity,including a pore-forming mechanism of crystal proteins,an inhibition-like mechanism of thuringiensin and a degradation mechanism of proteases and/or chitinases.Trojan horse mechanism from degradation mechanism of proteases exists Bs group,not just in B.nematocida,and degradation mechanism of chitinases were newly found in spore-forming Bacilli.120 spore-forming Bacilli across different families show diverse nematicidal capability,diverse nematicidal factors bring pathogenesis,necromeny and phoresy life styles to spore-forming Bacilli,indicating that spore-forming Bacilli is a good resource of nematicidal bacteria and studying relationship between bacterial pathogens and hosts.What’s more,phenomenon spore-forming Bacilli with different genetic backgroundsshow nematicidal activity and share similar virulence factors which contribute to their nematicidal capacity imply that different spore-forming Bacilli evolve nematicidal activity convergently in soil.Some spore-forming Bacilli showed high nematicidal activity in vitro,but have few nematicidal protein factors,demonstrating that secondary metabolites may contribute to this bacterial nematode pathogenesis.A database based on survey of nematicidal secondary metabolites from plant,fungi,bacteria and unclear organisms were set up and used as references of identifying the nematicidal secondary metabolites of spore-forming Bacilli.The metabolome showed that 120 nematicidal spore-forming Bacilli produced 734 secondary metabolites,including 513 non-peptide metabolites,174 tripeptides and 47 dipeptides.Comparison with the set up database by us and KEGG compound database,225 out of the 513 non-peptide metabolites have diverse bioactivities,including nine nematicidal compounds,selamectin,prometon,phenylacetic acid,diethyltoluamide,diethylcarbamazine,crotamiton,benzyl benzoate,benzyl alcohol and amodiaquine,and B.firmus produce the most,up to five of the above nine secondary metabolites.Tripeptides and dipeptides produced by nematicidal spore-forming Bacilli,especially cyclic dipeptides may play roles in the bacterial nematicidal process.Studying the biosynthesis of these secondary metabolites is helpful to mine spore-foriming Bacilli that containing these metabolites and to make products that control plants parasitic nematodes.B.thuringiensis,B.cereus,B.subtilis,B.pumilus,B.firmus,B.toyonensis,L.sphaericus,B.laterosporus and B.brevis were highly nematicidal species,and the rate of screening nematicidal spore-forming Bacilli from these species is higher than that of others,and the nematicidal activity is also relatively higher.Different combinations of nematicidal spore-forming Bacilli screened may be helpful to control plant parasitic nematodes because these strains may share similar nematicidal factors and mechanisms. |
