| Achnatherum inebrians,commonly known as drunken horse grass,is a perennial grass that is widely distributed in the northwest regions of China.Nearly 100% of naturally growing A.inebrians plants are host to a mutualistic Epichlo(?) fungal endophyte.Pseudomonas is the dominant bacterial group that can be isolated and cultured from the rhizosphere,phyllosphere and seed of A.inebrians.When A.inebrians plants were inoculated with a Pseudomonas strain isolated from the root system of this grass,it was found that the interaction between the Epichlo(?) endophytic fungus and the Pseudomonas promoted the seed germination and antioxidant enzyme activity.In this current study,Pseudomonas strains previously isolated from various parts of A.inebrians,including root,were used to assess intereactions with endophyte-free(EF)and endophyte-infected(EI)A.inebrians seeds.Through a germination test and greenhouse pot experiment,the differences of EI/EF seed germination,seedling growth,physiological and biochemical responses,and nutritional quality of plants inoculated with the inoculated bacteria strains were systematically compared,and the Pseudomonas strains that had significant promotion and inhibition effects on the growth of the drunken horse grass were selected for whole genome sequencing.The sequencing results were analyzed pan-genome.The main results of the effect of Pseudomonas on the growth and molecular mechanism of A.inebrians are as follows:1.The endophytic fungus had significant(P<0.05)effects on germination potential,germination index,seedling length and fresh weight.Inoculation of Pseudomonas strains(St1-St16 and St21-St29)had significant(P<0.05)effects on seed germination percentage,germination potential,germination index,root length,seedling length,fresh weight and dry weight.According to the membership function,30 of the 34 Pseudomonas strains had promoting effects,and 4 had inhibiting effects,on seed germination.The results of germination test and membership function analysis showed that different strains of the same Pseudomonas species(Sp1-Sp12)had the same promoting or inhibiting effects on the germination and growth of A.inebrians.Both the endophytic fungus and Pseudomonas had significant(P<0.05)effects on tiller number,fresh weight and dry weight;endophytic fungi,Pseudomonas and their interaction had significant(P<0.05)effects on plant height and the number of reproductive tillers.2.Compared with the liquid medium control(LBCK)and sterile water control(WCK),inoculation with Sp2 and Sp5 significantly(P<0.05)increased the photosynthetic rate,stomatal conductance and transpiration rate of A.inebrians;inoculation with Sp11 strain significant(P<0.05)decreased the photosynthetic rate of the leaves of A.inebrians,but significantly(P<0.05)increased the intercellular carbon dioxide concentration of leaves.Compared with LBCK and WCK,inoculation with other strains,except Sp1,Sp2 and Sp3,significantly(P<0.05)increased the content of total nitrogen in the leaves of A.inebrians.The total potassium content of EF leaves inoculated with strain Sp3 was significant(P<0.05)higher than CK,but the total potassium content in EI leaves was significant(P<0.05)lower than the control.Inoculation with strains Sp3,Sp9 and Sp10 significantly increased(P<0.05)total phosphorus content in the leaves of A.inebrians;and inoculation with all strains significantly(P<0.05)increased the total sodium content of the leaves.The organic carbon content of EI leaves inoculated with strains Sp1,Sp2,Sp3 and Sp5 was significantly(P<0.05)higher than of EF leaves.Compared with LBCK and WCK,the strains Sp1-Sp12 significantly(P<0.05)increased the content of IAA,ABA,BR,Ct K,SA and JA of leaves;All strains except Sp1 significantly(P<0.05)increased the content of GA in the leaves.Compared with LBCK and WCK,inoculation with strains Sp4,Sp5,Sp8,Sp9,Sp10 and Sp11 significantly(P<0.05)increased crude protein content in leaves;The crude fiber content of Sp5 and Sp6 EF inoculated leaves was significantly(P<0.05)lower than that of the control,and the crude fiber content of Sp10,Sp11 and Sp12 EI leaves were significant(P<0.05)lower than the control;The crude fat content of leaves of plants inoculated with strain Sp2 was significantly(P<0.05)higher than the control.3.The results of whole genome sequencing and pangenomic analysis showed that the GC content of the 7 Pseudomonas strains was between 60% and 63%.The total base number in descending order is Sp5,Sp1,Sp3,Sp12,Sp11,Sp7 and Sp10.The average total base number of the four strains Sp1,Sp3,Sp5 and Sp7 which significantly promoted the growth and metabolism of A.inebrians(283675829 bp)was much smaller than that of the three strains Sp10,Sp11 and Sp12 which significantly inhibited the growth and metabolism of A.inebrians(39332457 bp).The functional annotation results of COG(clusters of orthologous groups)genes in the genomic map of the 7 strains were mainly for amino acid transport and metabolism,general function prediction,energy production and conservation,inorganic ion transport and metabolism,and transcription.The number of core gene families of Sp1,Sp3,Sp5 and Sp7(2291,accounting for 26% of the total pangenomic gene family)was much smaller than that of the core gene family of the inhibiting strains Sp10,Sp11 and Sp12(4722,accounting for 99% of the total pangenomic gene family).The functional annotation information of the core gene family,non-core gene family and strain specific gene family was annotated to the ABC transporter.Phylogenetic tree analysis and collinearity results show that Sp10,Sp11,and Sp12 are closely related,and six identical or similar genomic islands-Genomic＿island＿1,Genomic＿island＿2,Genomic＿island＿3,Genomic＿island＿5,and Genomic＿island＿6 and Genomic＿island＿8-were found in the three strains,comprising 151 genes.Fifty-two of the 151 genes can be annotated to the 49 metabolic pathways available in the KEGG database,which mainly include the transposase IS5 family.In conclusion,this study involving the inoculation of A.inebrians with Pseudomonas,combined with seed germination and a greenhouse pot experiment,and analyzing seed germination,plant physiological,biochemistical and nutritional quality differences of EI and EF A.inebrians,found that the effects of Pseudomonas on A.inebrians seed germination and seedling growth were diverse,and this diversity was related to the differences of Pseudomonas gene composition and genomic island. |
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