Study On Diversity Of Endophytic Bacteria In Dominant Plants On Alpine Grassland In The East Qilian Mountains

The endophytic bacteria diversity of Gentiana macrophy Pall, Kobreasia capillifolia, Polygonum viviparum, Anaphalis lactea Maxin and Stipa purpurca Griseb, Which are the dominant plants on the typical alpine grasslands Polygonum grassland, Kobresia grassland, Grass grassland in the Eastern Qilian Mountains, China, were studied. Traditionally microbiological isolating culture, Physiological and biochemical method and molecular biology technologies such as PCR-RFLP, PCR-DGGE,16S rDNA sequence analysis, nifH sequence analysis have been used to study diversity of function(ability of nitrogen fixing, phosphorus-solubilizing, ability to secrete IAA and to decompose cellulose), diversity of physiological and metabolic functions, genetic diversity and diversity of ecological distribution of the endophytic bacteria in the sample plants. Meanwhile, community diversity and dynamic condition of endophytic bacteria in the five sample plants have also been explored.The below are main results of the study:1. Research on functional diversity of the cultured endophytic bacteria11 strains nitrogen-fixing bacteria were screened from the 150 strains, and the 11 strains were Bacillus sp.(QD13, Z19, ZD1), Rhizobium sp. (LRC6), Variovorax sp. (LRD9) Acinetobacter sp. (Z21), Lysinibacillus sphaericus(Z22), Sphingomonadaceae sp.(ZC8), Agrobacterium sp. LZD29 and two uncategorized(QB6, QB11).7 strains were identified as phosphate-solubilizers and they were Bacillus sp. (QB23, RA5, RC3), Stenotrophomonas sp. (QB22), Paracoccus sp. (RA8) and two uncategorized (RD12, XA10).22 strains were found to have the ability to secrete IAA and they were Bacillus sp. (QB8,QB13,QB23, RA5, RC5, RC24, XB4, ZA12, ZHA9, ZHA14), Lysinibacillus sp. (ZD19) Promicromonospora sp. (LZHA4) and 10 uncategorized (QA3, XA10, XB3, ZC5, ZD15, ZD18, ZHA10, ZHA11, ZHB1, ZHC4).The 18 strains were identified as cellulose-digesters and they were Bacillus sp. (QB8, QB13, RC16, Z19, ZHA3, ZHA9) Lysinibacillus sp. (X5), Paenibacillus sp. (X6) and 10 uncategorized (QA7, QB26, QC13, RD7, RC26, ZA14, ZC5, Z17, Z20, ZHA10). Meanwhile some were found to have more than one function. Strain Bacillus sp. (ZD1) had the ability of nitrogen-fixing and secretion to IAA. Strains Bacillus sp. (QB23, RA5) were identified as phosphate-solubilizers and also had the ability to secrete IAA. Strains Bacillus sp. (QD13, Z19) had the ability of nitrogen-fixing and digesting cellulose. Strains Bacillus sp. (QB8, QB13, ZHA9), ZC5 and ZHA10 could secrete IAA and digest cellulose. All the above showed that the cultured endophytic bacteria under alpine plants had rich functional diversity.Mechanism of phosphorus-solubilization of strain RA8 was explored and ability to phosphorus-solubilize phosphorus in soil and promote maize growth were also determined. Phosphorus-solubilizing quantity of RA8 in PKO medium was 315.36 mg/L and some acid getting from metabolism of RA8 could phosphorus-solubilize some hard ones, although which were not the main mechanism of phosphorus-solubilization of strain RA8. Phosphorus-solubilizing quantity of RA8 in soil without sterilization was 115.53 mg/L and quantity in soil with sterilization was 77.60 mg/L. RA8 could made help in the growth of maize and could improve plant height and plant weight significantly.Optimizations of culture condition of strain LZHA4 and strain X5 were made, and LZHA4 was the strain that could secrete the most IAA and strain X5 was the strain that could decompose the most cellulose. The quantity of IAA that LZHA4 secrete in medium King+ glucose 6 g/L+ tryptophane 0.1 g/L was 129.68 mg/L and the quantity in medium King+ glucose 6 g/L was 118.94 mg/L. There wasn't much difference between the two medium and the best medium for LZHA4 secretion to IAA was King+ glucose 6 g/L in consideration of cost.The best medium for strain X5 producing cellulose was Peptone 5 g/L, yeast powder 5 g/L and sodium carboxymethylcellulose 5 g/L, and together with pH 8.5 and temperature 20℃. Enzymatic activity of cellulose in the condition was 0.862 U, which was the 1.89 times before the optimization.2. Research on genetic diversity of the cultured endophytic bacteria16Sr DNA gene sequence of 102 strains were determined in the research and got GenBank access numbers. nifH gene sequence of strains Azotobacter chroococcum 1.178,Bacillus sp. (Z7, Z19),Acinetobacter sp. (Z21),Lysinibacillus sphaericus (Z22) were got in the research and got the GenBank access numbers. According to the analysis on 16Sr DNA gene sequence, there are 57strains Bacillus in all the determined 102 strains, and the percentage is 55.9%, which showed that they were the dominant microbile. There were 7 strains Sphingopyxis and the percentage was 6.9%. There were 4 strains Serratia, and the percentage was 3.9%; 3 strains Acinetobacter, and the percentage was 2.9%.There were 3 strains Lysinibacillus, and the percentage was 2.9%.There were Arthrobacter,2 strains Curtobacterium,2 strains Pseudomonas,2 strains Sporosarcina,2 strains Staphylococcus, 2 strains Agrobacterium,2 strains Stenotrophomonas and 2 strains Variovorax.There was 1 strain Sphingobium,1 strain Sphingomonas,1 strain Exiguobacterium,1 strain Kocuria, 1 strain Micrococcus,1 strain Planomicrobium,1 strain Pedobacter,1 strain Paracoccus,1 strain Phyllobacterium,1 strain Paenibacillus,1 strain Sphingomonadaceae and 1 strain Promicromonospora. There were total 24 genus endophytic bacteria in the study and these were the first time that five of them:Paracoccus (RA8), Sphingobium (ZA7), Sphingopyxis (QC2, QC9, RA2, RB, RB3, RC1, ZD24), Lysinibacillus (X5, ZD16, Z22) and Planomicrobium (QD9) were proved to be endophytic bacteria in plants. All the above showed that endophytic bacteria in alpine plants have rich genetic diversity.3. Distributive diversity of cultured endophytic bacteria community in alpine plants.From the characteristic of cultured endophytic bacteria in alpine plants, there were six bacteria groups:α-proteobacteria,β-proteobacteria,γ-proteobacteria, HGC, LGC and CFB groups in the five alpine plant, and LGC group had the richest genus. There were 7 genus from LGC group, accounting for 63.7% in the total strains. From community structure of endophytic bacteria in different grassland types and different plants, endophytic bacteria community from different grassland types had the same type and difference between each other, and endophytic bacteria community from different plants on different types of grassland had higher similarity but specific bacteria community. For example, endophytic bacteria community in dominant plants Gentiana macrophy Pall(Gentianaceae) and Kobreasia capillifolia(Cyperaceae) on Kobresia grassland both hadγ-proteobacteria, HGC and LGC groups, whileα-proteobacteria was the specific groups in Gentiana macrophy Pall(Gentianaceae) and CFB was the specific groups in Kobreasia capillifolia(Cyperaceae). Endophytic bacteria community in dominant plants Anaphalis lactea Maxin(Asteraceae) and Polygonum viviparum(Polygonaceae) on Polygonum grassland hadα-,β-,γ-proteobacteria and LGC groups while HGC was the specific groups in Anaphalis lactea Maxin(Asteraceae). All the above showed that endophytic bacteria community had diversity of distribution in the plants and also showed that biological characteristics of host plants and entironment where the host plants lived had effect on the distribution of endophytic bacteria in the host plants.4. Distributive diversity of cultured endophytic bacteria in organs of alpine host plants The distribution of endophytic bacteria in the organs of alpine plants, such as root, stem, leaf and blossom, have rich diversity of entironment distribution in quantity and variety. There were 13 genus in Polygonum viviparum and 8 of them were in the root, which were the most one. There was only bacillus in the stem, which was the least one, and the blossom and leaves were in the middle. There were 9 genus in Anaphalis lactea Maxin and most of them were in the leaves and 6 of them were in the leaves. There were 2 genus in the blossom of Anaphalis lactea Maxin, which was the least one, and the root and stem were in the middle. There were 8 genus in Kobreasia capillifolia and 7 of them were in the root. There were 7 genus in Gentiana macrophy Pall and 4 of them were in the stem. There were 2 genus in the leaves and blossom of Gentiana macrophy Pall. There were 4 genus in Stipa purpurca Griseb and 3 of them were in the root. There was only bacillus in the leaves of Stipa purpurca Griseb.5. Diversity of physiological and metabolic functions10 sample strains bacillus, which were dominant genus, were chosen and determined their ability to utilize carbon (monosaccharide, disaccharide, organicacidsalt), nitrogen (inorganicnitrogen, organicnitrogen), and the macromolecule (starch, cellulose, glutin, casein). The result showed that the ability of 10 strains bacillus to utilize the material was difference, which showed rich diversity of physiological and metabolic functions. Bacillus sp.(ZC12, QB8), B. subtilis(QB23, Z19)and B.pumilus(ZA8)could utilize all the 4 tested monosaccharide. B. cereus (RA5, ZH4, ZH6) could only utilize glucose and Bacillus sp.(XA1) and B. spaericus (Z22) could utilize none of the 4 tested monosaccharide. B. cereus (RA5, ZH4) could utilize saccharose and B. cereus (ZH6) couldn't utilize saccharose. B. cereus (ZH4, ZH6) could utilize citrate and B. cereus (RA5) couldn't use citrate. B. subtilis (QB23, Z19), B. cereus (ZH4, ZH6, RA5), Bacillus sp.(ZC12, XA1) could deacidize nitrate while B. pumilus (ZA8), B. spaericus (Z22), Bacillus sp.(QB8) couldn't deacidize nitrate. Within the 10 strains, only Bacillus sp.(QB8) and B. cereus (RA5, ZH4, ZH6) could utilize tryptophan to produce indole. B. cereus (ZH4, ZH6, RA5), B. subtilis (QB23, Z19) and Bacillus sp.(ZC12, QB8) could hydrolyze starch while B. pumilus (ZA8), B. spaericus (Z22), Bacillus sp.(XA1) couldn't hydrolyze starch. B. subtilis (Z19) was the only one that could discompose cellulose and the other nine strains couldn't. Strain B. spaericus (Z22) was the only one that couldn't hydrolyze casein. All the above indicated that different strains of the same species and different species had rich diversity of physiological and metabolic functions.6. Analysis on dynamic states of endophytic bacteria in the five alpine plants using DGGENest-PCR amplification of total DNA of the alpine plants and DGGE method were used to analyze the dynamic state of endophytic bacteria in different period of plants. The results showed that Shannon-Wiener index (H) of endophytic bacteria in the five plants were 1.153-1.533 in July and 1.127-1.502 in September and there were difference in Shannon-Wiener index (H), Evenness (EH) and Richness (S) of endophytic bacteria in different periods of the same plants life. Shannon-Wiener index (H), Evenness (EH) and Richness (S) of endophytic bacteria in Kobreasia capillifolia, Polygonum viviparum, Stipa purpurca Griseb was higher in July than in September. Anaphalis lactea Maxin was on the contrast of the three, Shannon-Wiener index (H), Evenness (EH) and Richness (S) was higher in September than in July while endophytic bacteria in Gentiana macrophy Pall had the same in July and in September. The similarity of the population of endophytic bacteria in July and September of the five plants was lower and the similarity was less than 60 percent. There were less changes in the community of endophytic bacteria in July and September in Gentiana macrophy Pall and Anaphalis lactea Maxin, and the similarity was relatively high. There were more changes in the community of endophytic bacteria in July and September in Anaphalis lactea Maxin, Polygonum viviparum and Stipa purpurca Griseb. All the above showed that host plants and different periods in life had effect on the community of endophytic bacteria, which had rich diversity of community.7. Analysis on diversity of endophytic bacteria in Polygonum viviparum using PCR-RFLPTwo Restriction Enzymes HhaⅠand HaeⅢwere used to make RFLP analysis on 16S rDNA gene segments of endophytic bacteria in Polygonum viviparum and genetic sequence were made to the obviously different clone. The results showed that there were 7 endophytic bacteria groups in Polygonum viviparum and they wereα-proteobacteria,β-proteobacteria,γ-proteobacteria,δ-proteobacteria, HGC, LGC and CFB groups, furthermore,γ-proteobacteria groups had the highest percentage 31.3% in the cloned samples. The percentage ofβ-proteobacteria groups in the cloned samples was 25%,α-proteobacteria groups was 15.6%, CFB groups was 12.5%, LGC groups was 9.4%,δ-proteobacteria groups was 3.1%, and HGC groups was 3.1%. Compared with cultured endophytic bacteria in Polygonum viviparum, much more endophytic bacteria groups have been identified using uncultured method, and the genetic information of the three extra endophytic bacteria groups:δ-proteobacteria, HGC and CFB groups have also been found. Besides16S rDNA gene segments of endophytic bacteria have been amplified from the total DNA of the plants Gentiana macrophy Pall, Kobreasia capillifolia, Polygonum viviparum, Anaphalis lactea Maxin and Stipa purpurca Griseb using primer 799f-1492r, which showed that primer 799f-1492r could remove the interference from DNA of chloroplastid and mitochondrion of the host plants and it could be widely used in the research of uncultured method of endophytic bacteria in many plants.Through study on diversity of endophytic bacteria in dominant alpine plants, it had been proved that there were plenty of cultured and uncultured bacteria species in alpine plants and the endophytic bacteria had rich diversity of physiological and metabolic functions, genetic diversity and functional diversity. Therefore, the study provided theoretical basis and fundamental research for the exploration and utilization of endophytic bacteria in alpine plants, which had great potential and need to be explored further in the future.