Screening And Inhibition Mechanism Of Strain ST4 On Sexual Mating Of Sporisorium Scitamineum

Sugarcane smut is a fungal disease caused by S.scitamineum,which can cause severe economic losses in sugarcane industry,causing 5 billionseconomic losses every year.Infection depends on the mating of bipolar sporida to form a dikaryon and develops into hyphae to penetrate the meristematic tissue of sugarcane.In this study,we set to isolate bacterial strains capable of blocking the fungal mating and evaluate their potential in control of sugarcane smut disease.The main results are as follows:1.More than 90 rhizosphere soilsamples were collected from Guangdong,Guangxi,Jiangxi,Xinjiang,Henan and Hainan in China,from whichmore than 8 thousands bacteria with morphologically different characteristics wereisolated,amongst,118 strainsdisplayed potent inhibitory activity against the mating of S.scitamineum.After morphological observation,10 different species with high inhibitory activity were selected,including 8 species of bacteria and 2 species of actinomycetes.Isolate ST4,a Gram-negative bacteriumfrom rhizosphere in Shantou city of Guangdong Province,was selected for further study.Phylogenetic analyses,microscopy,biochemical characterization,molecular biology identification,and literature description showed that the isolate was most similar to Pseudomonas guariconensis.2.In order to further study the inhibitory mechanism of strainST4 on the sexual mating of S.scitamineum,and to facilitate the subsequent gene function analysis,genome sequencing of P.guariconensis ST4 was performed in this study.Results showed that the genome of strainST4 in a chromosome with 5135208 bp in size,63.57% of GC content,containing 4576 genes covering 4475352 bp with an average length of 978 bp and coverage of 87.15%.Number of predicted tandem repeats,small satellite sequences and microsatellite sequences was 216,of which 124 tandem repeats with a total length of 26925 bp ranging from 3 to 1605 bp,covering 0.5243% of the genome;83 minisatellite sequences and 9 microsatellite sequences;73 tRNAs,19 rRNAs and 15 sRNAs.Gene function annotation showed that there are 13 genes involved in interaction of signaling molecules,of which 3 ere related to insect toxins,possiblyassociated withfungalinhibitory activity of strainST4 observed in this study.3.A total of 40 thousandsof strain ST4 mutants were obtained by Tn5 transposoninsertion mutagenesis,amongst,109 lose inhibitory activity on the sexual mating of S.scitamineum.From results of flanking sequencing of the Tn5 insertion sites after hiTAIL-PCR amplification,35 genes related to pyrroloquinoline quinone synthesis,glucose dehydrogenase,and 2-isopropyl malic synthase were identified.4.After ethyl acetate extraction,silica gel and HPLC separation,and tracking activity,we found that metabolites of strainST4 were rich and diversifiedin antibacterial active ingredients,some of which can both inhibit the sexual mating and the growth of hyphae such as ST4-2-8;and some can only inhibit the sexual mating(ST4-2-7-5)or the growth(ST4-2-4 and ST4-2-6).Analysis by NMR and LC-MS identified ST4-2-8 as indole derivatives of indole-3-formaldehyde,which inhibited the sexual mating at a low concentration,and the growth ata high concentration,with up to 94% of control rate on potted corn smut disease.5.Optimization of ST4 culture conditions showed thatST4 fermentantation broth produced more inhibitory ingredients under pH6.0,and glucose was essential for ST4 antibacterial effect,with an optimum concentration of 6%;oxygen played a key rolein bacterial culture.The higher the speed,the better antibacterial activity of the fermentation liquid;in addition,Mg ion was essential for the growth of ST4,and addition of appropriate metal ions helped to produce ST4 antibacterial substances.6.Pathogenisis test showed that injection of sugarcane in pot trial using bipolar sporida mixture in addition of glucose and ST4 got a least disease rate of 13.04%,64.74% lower than that using only sporida mixture,with a good control effect of 83.23%,but got poor and unstable control effect in field trial.Generally speaking,ST4 can not only affect the growth of bipolar sporida and the dikaryotic hyphae,but also inhibit the mating process of sporesin different cell lines.The specific inhibition mechanismswere possibly as follows: metabolites of ST4 causedamage onthe morphology of biopolar cells,change their affinity from mutual compatible into incompatible,thereby inhibit the occurrence of sexual mating;since the metabolites produced by ST4 are complicated and diversified,different metabolites may play different roles,some inhibit the growth of spores and hyphae,some affect the affinity of the spores,which needs further in-depth study to unveil the corresponding inhibitory mechanisms.