| Bacillus amyloliquefaciens SQR9, isolated from the rhizosphere of cucumber, can control Fusarium wilt of cucumber. The strain was proved to be an outstanding plant growth-promoting rhizobacteria (PGPR) strain by its ability to inhibit the growth of Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FOC) and decrease the incidence of Fusarium wilt in field experiments, and a bioorganic fertilizer made of organic fertilizer fermented with B. amyloliquefaciens SQR9 is widely used in agricultural production.In this study, SQR9 showed an impressive plant-growth-promoting activity in greenhouse experiments. We detected factors which were involved in the plant-growth-promoting effects of SQR9, and investigated the IAA synthesis pathways in SQR9 through transcription analysis, gene knockout, gene fusion expression and transposon insertional mutant library, the main results abstained were summarized as follows.1. Greenhouse experiments showed that B. amyloliquefaciens SQR9 significantly promoted growth of cucumber and maize. To avoid the influence of bacteria cells on seedlings, equal amount of inactivated B. amyloliquefaciens SQR9 cells were used as control (CK). Obvious differences between CK replications were not observed. When 5* 108CFU B. amyloliquefaciens SQR9 suspensions were applied to each pot (T1 treatment), the cucumber biomass weight, shoot height, root length and root surface area increased by 60.1%,45.7%,29.3% and 30.7%, respectively. When 1×109 CFU cell suspensions were applied (T2 treatment), increases amounted to 90.0%,71.6%,56.3% and 65.6%, respectively. For maize, in T1 treatment biomass, shoot height, root length and root surface area increased by 36.2%,30.6%,32.1% and 20.0%, and 65.3%,47.8%,42.6% and 52.0% of T2, respectively.2. To study if volatile chemicals produced by B. amyloliquefaciens SQR9 could affect plant growth, Arabidopsis thaliana (ecotypes Columbia) was inoculated with SQR9, then Escherichia coli DH5a and PBS served as controls. After 16 days incubation, B. amyloliquefaciens SQR9 significantly stimulated A. thaliana growth compared with the controls.3. Chemical analysis showed that B. amyloliquefaciens SQR9 could produce acetoin,2, 3-butanediol, phytase, IAA and GA3 in laboratory. And addition of L-tryptophan in Landy media increased the yield of IAA, suggested that IAA production in SQR9 is tryptophan dependent.4. Based on the proposed IAA biosynthesis pathways in both plants and bacteria, the SQR9 genome (NCBI accession No. CP006890) was mined to screen the candidate IAA synthesis genes according to their deduced protein domains, which showed putative enzyme activities already known in IAA metabolism. Because all the gene candidates were potentially involved in the tryptophan-dependent IAA biosynthesis, the active genes were identified from the transcriptional responses of the selected genes to the addition of tryptophan. Six of the genes were found to be significantly induced by tryptophan:patB, encoding a conserved hypothetical protein predicted as an aminotransferase, increased 3.5-fold; yhcλ, a predicted nitrilase, increased 3-fold; dhaS, indole 3-acetaldehyde dehydrogenase, increased 2.5-fold; ysnE, a predicted tryptophan acetyltransferase, increased 2-fold; yclB, encoding an aromatic-acid decarboxylase, increased 1.5-fold; and yclC, located in the same operon as yclB, encoding a UbiD family decarboxylase, increased 2-fold. These six genes were proposed to be involved in the tryptophan-dependent IPyA, TAM, and IAN pathways, as well as an uncharacterized IAA biosynthesis pathway.5. To confirm their contributions to IAA production by B. amyloliquefaciens SQR9, these six genes were individually deleted. Quantification of the IAA amounts present in culture filtrates of gene knockout mutant strains showed that strains AysnE, AdhaS, AyclC, and AyhcX formed only 14%,23%,45%, and 76% of IAA produced by the wild type SQR9, respectively, whereas ApatB and AycIB mutant strains showed similar IAA production with wild-type SQR9. Complementation of the four IAA production reduced mutants with the B. amyloliquefaciens-E. coli shuttle vector pUBC19 carrying the corresponding deleted genes restored the IAA production. These results suggested that the pathway including tryptophan acetyltransferase was the main route for IAA biosynthesis in SQR9. The IPyA and TAM pathways also contributed substantially, but the IAN pathway only slightly contributed to IAA production in SQR9.6. From the above results, genes constituted a possible entire IPyA pathway including patB, yclC and dhaS were identified in SQR9. To verify the IPyA pathway in SQR9, these three genes were tandemly cloned into the Bacillus-E. coli shuttle vector pUBC19 under the control of the P43 promoter and then homologously and heterologously expressed in strain SQR9 and B. subtilis 168. Homologous expression of the IPyA pathway in B. amyloliquefaciens SQR9 (strain SQR9-E) resulted in nearly 4 times the IAA production (42 mg l-1) of the control strain SQR9-CK, and B. subtilis 168 carrying the IPyA pathway genes (strain 168-E) produced approximately 22 mg l-1 IAA,10 times as much as the control strain 168-CK.7. A shuttle plasmid pMarA, which contains a mariner-based transposon, was used to construct a random insertion library for screening mutant strains, which shows variation in IAA production. Three mutants showing stable change in IAA production were selected from 3,000 mutants. Low IAA was detected in the culture supernatant of SQR9-IAA1 strain, and IAA production of SQR9-IAA2 was nearly 50% of wild type, while in SQR9-IAA3, IAA production was increased by nearly 240% compared with that of wild type strain. The roles of these three genes in IAA production need further investigation.8. To study the effect of different IAA production strains of SQR9 on the growth promoting ability, strains that showed variation in IAA production were applied to this part. The cucumber root was mostly stimulated at the concentration of 107 CFU ml’1 of wild type SQR9 strain. In this cell concentration, the root length and root surface area increased by 137% and 88% in SQR9 treatment as compared with control. Strain AysnE, which impaired in IAA production showed no significant difference with control. The root length and root surface area were increased by 58% and 166% in IAA over production strain SQR9-E treatment as compared with control, but decreased by 32% and 15% as compared with SQR9 treatment. Effects of wild type B. amyloliquefaciens SQR9 and its IAA production variation strains on cucumber seedlings growth regarding the biomass weight and shoot height and all of the four strains have growth-promoting effects compared with CK. Strain SQR9-IAA1 treatment was impaired in its capability to support plant growth, the biomass weight and shoot height of cucumber were decreased by 24% and 19% as compared to the wild type SQR9 treatment. And that of maize decreased by 21% and 17%. The plant growth effect of SQR9-IAA2 showed similarity with wild-type strain. In AysnE treatment, the biomass weight and shoot height of cucumber were decreased by 23% and 15%, and 15% and 16% in maize as compared to SQR9 treatment. SQR9-IAA3 and SQR9-E strains, which produced more IAA than the wild type also showed more significant growth promotion of cucumber and maize over that of other strains. The biomass weight and shoot height of cucumber were increased by 40% and 19% in SQR9-IAA3 treatment,41% and 18% in SQR9-E treatment. The biomass weight and shoot height of maize were increased by 21% and 29% in SQR9-IAA3 treatment,18% and 31% in SQR9-E treatment. |
PDF Full Text Request