Font Size: a A A

Isolation, Identification And Back Inoculation Of Endophytic Moderately Halophilic Bacteria Of Halophyte Suaeda Salsa L. In Seaside Saline Soil Of Dongying, China

Posted on:2011-06-12Degree:MasterType:Thesis
Country:ChinaCandidate:C X CuiFull Text:PDF
GTID:2120360308465561Subject:Microbiology
Abstract/Summary:PDF Full Text Request
Suaeda salsa L. generally grows in saline soil, hypersaline soil, lakeside, river bank and seaside, which is the typical plant of saline alkali soil. S.salsa L. is a salt-dilution halophyte. It has powerful ability to dilute salt so that the total salt content in S.salsa L. is twice that in surrounding environment. S.salsa L. is an important salt tolerance model plant after Thellungiella halophila. It is also the nature habitat of moderately halophilic bacteria not studied.The traditional isolation method and molecular biological method were used to investigate the phylogenetic diversity of endophytic moderately halophilic bacteria of S.salsa L. in Dongying. And then a new strain isolated from S.salsa L. was subjected to a polyphasic taxonomic study. Back inoculation test of an isolate was performed last. The main conclusions of this paper are as follows:1. The study in endophytic bacteria of S.salsa L. was reported for the first time. 15 different endophytic bacterial strains were isolated from S.salsa L. in seaside saline soil of Dongying. Among these isolates 14 Gram-negative strains grew best in 3%–7% (w/v) NaCl belonging to moderately halophilic bacteria. The Gram-positive salt-tolerant Bacillus ST306 was able to grow in 13% (w/v) NaCl. Physiological and biochemical tests for the isolates were performed. 16S rRNA gene sequences of the isolates were amplified and submitted to GenBank. 16S rRNA gene sequences similarities of five isolates with respect to the known bacterial species were < 98%, which indicated that there was some novel bacteria species existed in S.salsa L.. Phylogenetic trees were constructed with the aid of the MEGA 4.0 software package using the neighbour-joining algorithms. 2. According to physiological and biochemical characteristics, 16S rRNA gene sequences and phylogenetic analysis, 15 isolated endophytic bacterial strains were classified into four groups, belonging to genera Chromohalobacter, Kushneria, Halomonas or Bacillus separately. 16S rRNA gene sequences of 4 strains in Group I had the highest similarity to Chromohalobacter israelensis (95%). Group II which contained 7 strains of the genus Kushneria is the dominant group in S.salsa L.. The strains in Group III bear the closest affiliation to Haererehalobacter sp. JG 11 (99%), which is not classified definitely. The only endospore-forming bacterium was classified into group IV. Its 16S rRNA gene sequence similarities with respect to the known Bacillus species were≤96%, which was presumed to be potential novel species. The research results indicated that there was not only abundant phylogenetic diversity of moderately halophilic bacteria, but also some unknown bacteria groups existed in S. salsa L.. Among these isolated strains, three strains produced proteinase; 14 strains produced esterase; 8 strains produced DNase; 11 strains produced galactosidase; 14 strains produced urease. The results supplied kinds of meterials for new biological catalyzer research.3. One moderately halophilic, Gram-negative bacterium isolated from halophyte S.salsa L. in seaside saline soil of Dongying, China, was subjected to a polyphasic taxonomic study. The phenotypic, genotypic and phylogenetic characteristics of the strain matched those described for the genus Chromohalobacter. Strain ST307T was aerobic, motile and orange-yellow pigmented. The organism grew at NaCl concentrations in the range of 0.6%–20% (w/v), at 5–45oC and at pH 5–9. To determinate the DNA G + C content of genus Chromohalobacer, reversed-phase high performance liquid chromatography was used for the first time. Various bases were separated on a Venusil MP C18 column by using 90% double distilled water and 10% methanol as mobile phase. The G + C content of strain ST307T was 60.5 mol%, which is within the range described for the genus Chromohalobacter. On the basis of 16S rRNA gene sequence analysis, strain ST307T was found to be closely related to C.israelensis ATCC 43985T and Chromohalobacter marismortui GSP58. DNA–DNA hybridization studied by denaturalization speed in liquid phasic between strain ST307T and C.marismortui ATCC 17056T showed 28.1% relatedness. Therefore, the phenotypic, genotypic and phylogenetic results demonstrated that strain ST307T represents a novel species. The name Chromohalobacter Tungyingensis sp. nov. is proposed, with strain ST307T (= CGMCC 1.8902 = DSM 22428) as the type strain. Method and platform was established for describing new species in our laboratory through identification of strain ST307T.4. Back inoculation test of the isolated strain ST307T was performed. Dilute bacteria solution of strain ST307T was poured on the test S.salsa L. plants after them sprouting with the aim of infecting the plants. Endophytic bacteria were isolated from the test plants with 4–5 euphylla. Only one strain was isolated and named Y01. The similarity between 16S rRNA gene sequences of strain Y01 and ST307T was 100%. 6 random primers including S302, S307, S309, S344, S345 and S346 were selected for random amplified polymorphic DNA analysis on strain Y01 and ST307T because they showed better polymorphism. Random amplified polymorphic DNA fingerprints amplified by 6 random primers on gene DNA of the two strains were the same. Therefore, the results above demonstrated that strain Y01 and ST307T was the same one. Strain Y01 was just ST307T infection in test plants. Strain ST307T was indeed the endophytic bacterium of S.salsa L..
Keywords/Search Tags:Moderately halophilic bacteria, Chromohalobacter, Suaeda salsa L., Endophytic bacteria, Phylogenetic diversity
PDF Full Text Request
Related items