| Alfalfa(Medicago sativa L.),known as the“king of pasture”,is widely planted all over the world and is an important part of animal husbandry production.Disease has emerged as one of the important factors restricting the high quality and high yield of alfalfa.A large number of plant or animal pathogenic bacteria colonize the alfalfa seeds for a long time,and they can spread over a long distance with frequent seed trade.These“dangerous hitchhikers”will result in greater health risks for many hosts at the“grass-livestock”interface in the grassland agro-ecosystem.It is worth noting that Pantoea species colonized in alfalfa seeds are potential cross-kingdom pathogenic bacteria,they have the ability to infect plants and animals or humans at the same time.There is no doubt that the spread of these pathogenic bacteria in grassland agro-ecosystems poses a major threat to foraging grass,livestock,and human health.Therefore,the study on the pathogenicity and mechanism of cross-kingdom pathogenic bacteria is helpful to reveal the risk of infection and provides the theoretical basis for scientific prevention and control.In this study,a Pantoea species was isolated from alfalfa seeds,and its taxonomic status at the“species”level was determined based on the whole genome sequence.Alfalfa plants and SPF grade KM mice were used as experimental materials,and cross-kingdom pathogenic bacteria Erwinia persicina Cp2 was used as the control species.The pathogenicity of the Pantoea isolates was evaluated by constructing a plant root infection model and an animal intragastric infection model.Then,the virulence factors and pathogenic mechanism of the Pantoea isolates were explored by pan-genome analysis,comparative genomics,and transcriptome.The main results are as follows:(1)The dominant endophytic bacteria species in alfalfa seeds cultured by coating plate method was a nearly round isolate with a yellow single colony(names CQ10),and the number accounted for about 30%of the cultivable total bacteria.Based on phenotypic identification and16S r RNA gene sequence analyses,the strain CQ10 was identified as Pantoea species in the Erwiniaceae,Proteobacteria;Phylogenetic analysis of strain CQ10 based on the whole genome sequence showed that its related species were P.agglomerans and P.vagans,however the ANI and d DDH values between strain CQ10 and valid Pantoea species were lower than the corresponding species thresholds.Furthermore,we identified the physiological and biochemical characteristics of strain CQ10 by API 20E and Biolog GENⅢsystem.Based on all the above identification results,strain CQ10 should be attributed to a novel species within the genus Pantoea,for which the name Pantoea alfalfae sp.nov.is proposed.(2)The alfalfa plants were inoculated near the root with the strain CQ10 suspension(10~9CFU/m L).Starting from 10d,we observed that leaf spots began to develop on alfalfa leaves in CQ10 and Cp2 groups.With the increase of infection time,the number and area of leaf spots increased continuously.At 21 days,most leaves were completely yellowed and withered.In addition,the root growth of alfalfa was severely inhibited,and the inhibitory effect of strain CQ10 on roots was stronger than that of control strain Cp2.At 21 days after inoculation with bacterial suspension,strain CQ10 significantly inhibited the biomass and photosynthesis of alfalfa;The activities of antioxidant enzymes in roots,stems and leaves of alfalfa showed an upward trend,while the activities of defense enzymes showed a downward trend,while the soluble sugar and soluble protein showed an opposite trend.At the same time,the cell structure of root and stem cortex,palisade tissue,and sponge tissue in leaves of alfalfa in CQ10 and Cp2group were damaged and destroyed,and the effect of strain CQ10 was greater than that of control strain Cp2.Taken together,P.alfalfae sp.nov.CQ10 is a new pathogen of alfalfa plants,leaf damage and root inhibition are the main symptoms.(3)Mice were inoculated by orogastric gavage with strain CQ10 suspension.After inoculation,compared with the CK group,mice in CQ10 group and Cp2 group were dispirited and showed loss of appetite and immobility.Furthermore,strain CQ10 had a significant effect on the stomach coefficient and spleen coefficient of mice,which was consistent with that of control strain Cp2.At the same time,leukocytes,neutrophils,and monocytes in CQ10 and Cp2groups were higher than those in the CK group at 24 h and 48 h after gavage,but they all showed a downward trend after 48 h,the red blood cells and hemoglobin also showed a downward trend after 48 h.The pathological features of pathological sections were as follows:the main pathological changes of the heart in the bacterial treatment group were myocardial fibrinolysis and rupture,steatosis of myocardial cells,and adipocyte infiltration,and the symptoms worsened after 48 hours;The pathological changes of lung tissue were exfoliation of bronchial epithelial cells and infiltration of inflammatory cells,and the lung tissue injury was more serious at 24 h after gavage,and the degree of lung tissue injury became lighter with the increase of infection time;The main pathological changes of liver tissue were hepatocyte edema and fatty degeneration,which were the most serious at 24 h and 48 h,and alleviated after 72 h;The main lesions of gastric tissue were epithelial cells shedding and abnormal glandular structure,and the symptoms became worse with the increase of infection time,and the symptoms in CQ10group were more serious than those in Cp2 group.The main lesion of spleen tissue was neutrophil infiltration,and with the increase of infection time,the symptoms were more obvious;The main lesions of renal tissue were glomerular atrophy lobulation telangiectasia and brush edge injury of renal tubular epithelial cells.Thus,P.alfalfae sp.nov.CQ10 is pathogenic in mice.(4)The genome of strain CQ10 carried 563 virulence genes,which mainly function as nutritional/metabolic factors,motility,and immune regulation.Strain CQ10 also encodes type VI secretion system and siderophores involved in iron acquisition.The pan-genome analysis further revealed that compared with seven Pantoea species,the unique genes of strain CQ10were mainly involved in inorganic ion transport and metabolism,carbohydrate transport and metabolism,secondary metabolites biosynthesis,transport and metabolism,and cell motility.At the same time,comparative genomics revealed the relationship between strain CQ10 and cross-kingdom pathogenic bacteria E.persicina and P.aeruginosa,there are 77 common virulence factors in the three species,which are mainly involved in nutritional/metabolic factors,exotoxins,and adhesion processes;There are 12 unique virulence factors in strain CQ10,which are mainly involved in the processes of adhesion,biofilm formation,infection promotion,immune escape,and stress survival.To sum up,strain CQ10 possesses a variety of virulence factors,and its functions are mainly embodied in motility,adhesion,biofilm,iron acquisition system,and type VI secretion system.(5)Transcriptome analysis showed that there were 2,338 differentially expressed genes(DEGs)of strain CQ10 in the alfalfa host,including 1,065 upregulated genes and 1,273downregulated genes.There are 43 DEGs in mice,including 38 upregulated genes and 5downregulated genes.Among them,20 DEGs were co-expressed by strain CQ10 in two hosts.These DEGs are mainly involved in nutrient/metabolic factors,immunity,motility,adhesion,effector secretion system,and biofilm processes.Further analysis showed that Bap,a virulence factor involved in biofilm formation and adhesion,and NAP,a virulence factor involved in immune regulation,were differentially expressed by strain CQ10 in alfalfa and mouse hosts.It is worth noting that Bap is also differentially expressed when the control strain Cp2 infects alfalfa and mouse hosts,which indicates that biofilm formation is very important for the infection of these two cross-kingdom pathogenic bacteria.In addition,when analyzing the function of DEGs in the PHI-base database,it was found that pathogenic factors Leu B and Dps(atu2477)also played an important role in the infection of CQ10 and Cp2,among which Leu B was related to leucine biosynthesis and mainly regulated the expression of virulence factors such as iron metabolism,fimbriae formation,chemotaxis,and type VI secretion system.Dps(atu2477)mainly regulates iron homeostasis in bacteria.Therefore,the strategy adopted by strain CQ10 in cross-kingdom pathogenesis is to use virulence factors such as biofilm,motility,iron acquisition,and type VI secretion system to achieve host adhesion,colonization,immune regulation,and infection,among which biofilm formation mediated by virulence factor Bap may be very important for successful infection of strain CQ10.This study demonstrated for the first time that P.alfalfale sp.nov.CQ10,a new species of Pantoea,has the ability to co-infect and cause diseases across plant kingdoms and animal kingdoms.On this basis,the diverse virulence attributes of the strain CQ10 were identified by pan-genomics,comparative genomics,and transcriptomics.Furthermore,the cross-kingdom pathogenesis mechanism of the strain CQ10 was preliminarily explored.The obtained gene resources laid a solid foundation for the subsequent functional verification of virulence factors.The results of this study provide new insights for the follow-up study on the pathogenic risks of pathogenic bacteria to animal and plant hosts in grassland agro-ecosystem. |
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