However, as pH decreased, Cd removal efficiency in tests without nitrate addition was relatively higher than in tests with nitrate addition, especially at low pH conditions. This indicates that the removal capacity of iron hydroxide or iron oxide derived from the reaction between nZVI and nitrate is much lower than that of pristine nZVI. As seen in Fig. 3, the removal efficiency in both reaction systems increased by more than 20% when pH increased from 7.5 to 8.0, and 15% as pH increased from 8.0 to 8.5. Meanwhile, in a control test a significant increase in Cd removal efficiency was observed at pH 8.5 , although Cd started to precipitate out from water at pH 8.0 . This meant the increase in Cd removal efficiency was mainly due to the presence of nZVI. The sharp increase in removal efficiency and capacity may be due to three main reasons: the critical pH value for Cd hydrolysis þ and Cd23 þ) and precipitation 2) is 8.0 , which may have contributed to the increased removal rate through electrostatic interaction and deposition; the isoelectric point of nZVI is around 8.1 , below which nZVI particles are positively charged and above which they are negatively charged. The negatively charged surface at pH > IEP favors Cd adsorption due to strong electrostatic attraction. Thus, the change of surface charge of nZVI may also have contributed to the increased removal of Cd; and high pH, especially above 7, could also improve Cd adsorption on iron oxide . Comparing the results of these two series of experiments, the enhanced Cd removal capacity in the presence of nitrate most likely resulted from elevated solution pH. Specific sorption may have also contributed to Cd removal because of the relatively high removal efficiency observed around pH 6 .Under global warming and climate change,25 liter pot cultivated plants are encountering increased biotic and abiotic stresses, which lead to reductions of plant growth and reproduction and consequently economic losses. The use of plant endophytic bacteria to promote plant growth and increase tolerance of environmental stresses has provided an alternative to standard agricultural practices that has fewer safety concerns.
Endophytic bacteria can be defined as non-pathogenic bacteria that colonize the interior of host plants and can be isolated from surface-sterilized plant tissues. These bacteria can obtain a constant nutrient supply from host plants by living inside the plants and having close contact with plant cells. The endophytic bacteria colonization process is usually initiated at wounds and cracks in the roots by a rhizospheric population of the bacteria in the soil. After entering the plant roots, endophytic bacteria can systemically colonize the above ground parts of plants, including stems and leaves.Endophytic bacteria communities include five main phyla. Proteobacteria is the most dominant phylum isolated from host plants, which includes α-, β-, and γ-Proteobacteria. Actinobacteria, Planctomycetes, Verrucomicrobia, and Acidobacteria are also commonly identified. The most frequently isolated bacteria genera are Bacillus, Burkholderia, Microbacterium, Micrococcus, Pantoea, Pseudomonas, and Stenotrophomonas, with the two major genera being Bacillus and Pseudomonas. Several factors affect the composition of endophytic bacteria populations, including plant growth conditions, plant age, types of analyzed plant tissues, soil contents, and other environmental factors. Endophytic bacteria can have several beneficial effects on host plants, such as promotion of plant growth and yield, increased resistance to plant pathogens , enhanced tolerance to abiotic stresses, elimination of soil pollutants through the facilitation of phytoremediation, and production of various metabolites with potential applications in agriculture, medicine, and industry. Some endophytic bacteria help host plants acquire increased amounts of limited resources from the environment. This can include enhancing the uptake of nitrogen, phosphorous, or iron by expressing nitrogenase, solubilizing precipitated phosphates, or producing iron-chelating agents in bacteria, respectively. Some endophytic plant-growth-promoting bacteria can increase host plants’ metabolism and nutrient accumulation by providing or regulating various plant hormones, including auxin, cytokinin, gibberellins, or ethylene.
Auxin and ethylene are the two major hormones that affect plant growth and development and that are involved in plant-endophytic bacteria interactions. In addition to these four hormones, several endophytes can utilize signaling pathways mediated by salicylic acid, jasmonic acid, and ethylene to initiate induced systemic resistance and protect host plants from phytopathogen infection. A number of endophytic bacteria can also produce various antibiotics, toxins, hydrolytic enzymes, and antimicrobial volatile organic compounds to limit pathogen infection. We previously isolated a plant endophytic bacterium, Burkholderia sp. strain 869T2, from surface-sterilized root tissues of vetiver grass. Strain 869T2 can also live within banana plants, in which it promoted growth and reduced Fusarium wilt disease occurrence. Genomic sequences of the strain 869T2 contain the gene for 1-aminocyclopropane-1-carboxylate deaminase, which may modulate host plant ethylene levels. Strain 869T2 also has genes related to the synthesis of pyrrolnitrin, which may function as a broad-spectrum anti-fungal agent, as well as dioxin-degradation-related genes. Furthermore, strain 869T2 can degrade the toxic dioxin congener 2,3,7,8-tetrachlorinated dibenzo-p-dioxin , mainly via its 2-haloacid dehalogenase. A recent study compared the genome sequences of 31 Burkholderia spp. and reclassified Burkholderia cenocepacia strain 869T2 as Burkholderia seminalis. We also compared the genome sequences of the strain 869T2 with those of five published B. seminalis strains: FL-5-4-10-S1-D7, FL-5-5-10-S1-D0, Bp9022, Bp8988, and TC3.4.2R3. The strain 869T2 shared 93–95% of its genome with the other five B. seminalis strains. Furthermore, strain 869T2 lacked several genetic loci that are important for human virulence. Based on the results of our analysis of the core genome phylogeny and whole-genome average nucleotide identity , strain 869T2 was classified as B. seminalis. B. seminalis is a member of the Burkholderia cepacia complex , which is a group of Gram-negative, aerobic, non-sporulating, rod-shaped bacteria. Bcc consists of opportunistic human pathogens that exist in patients suffering from cystic fibrosis as well as pathogens of many vegetables and fruits,25 liter plant pot such as onion and banana . Contrary to the pathogenic traits that led to their original discovery, some Bcc bacteria have ecologically beneficial interactions with host plants.
The plant endophytic bacterium B. seminalis strain TC3.4.2R3, isolated from sugarcane, can serve as a bio-control agent to reduce infections with Fusarium oxysporum and the cacao pathogens Moniliophthora perniciosa , Phytophthora citrophtora, P. capsici, and P. palmivoraas well as orchid necrosiscaused by Burkholderia gladioli through the production of pyochelin, a rhamnolipid, and other unidentified diffusible metabolites. Another strain of Burkholderia seminalis, strain R456 isolated from rice rhizosphere soils, decreased the occurrence of rice sheath blight disease caused by Rhizoctonia solani. Furthermore, Burkholderia seminalis strain ASB21 was found to be able to produce the plant hormone auxin, promote rice seedling growth, and reduce aluminum toxicity symptoms in host plants. Similarly, a Burkholderia seminalis strain isolated from Bangalore, India can produce indole acetic acid and enhance tomato seedling growth. Although it is known that Burkholderia seminalis belongs to the plant-growth-promoting rhizobacteria , only limited strains and their promoting abilities are well characterized. In this study, we examined the amounts of IAA produced by B. seminalis strain 869T2 in various growth conditions, detected the strain’s siderophore synthesis and phosphate solubilization abilities, and demonstrated its growth-promoting abilities in several leafy vegetables, including pak choi, lettuce, and amaranth. Indole acetic acid production was determined as described previously, with minor modifications. Bacterial cultures were grown on LB media containing 100 µg mL−1 of tryptophan with different pH levels and appropriate antibiotic for 48 h at selected temperatures . Bacteria were also cultured on M9 salt media at 30 ◦C for 48 h with 100 µg mL−1 of tryptophan and 2% of different kinds of sugar: glucose, fructose, or sucrose. Fully grown bacteria cultures were then centrifuged at 5000 rpm for 10 min, and the supernatant was passed through a syringe filter with a pore size of 0.2 µm to remove bacteria. The 500 µL of supernatant was mixed with 1 mL of the Salkowski reagent and incubated at room temperature for 25 min. Finally, the concentrations of IAA in the supernatants were determined by comparison of the absorbance measured at 530 nm with a standard curve of 0–100 µg mL−1 IAA.Various growth parameters of different plant species were measured at selected days, ranging from 14 to 80 days, after inoculation with strain 869T2. The fresh weight, dry weight, and length of leaves and roots as well as the width, number, and surface area of leaves were measured in harvested pak choi, lettuce, and Chinese amaranth as described previously. The fresh weight, length, number, and color of fruits of hot pepper and okra were recorded following previously described methods. The chlorophyll content of the lettuce leaves was measured using a previously published protocol. Chlorophyll was extracted from the leaves with N, N-Dimethylformamide for 1 hour in the dark, and chlorophyll a and b concentrations were calculated from the absorbance of the crude extract at 647 and 664 nm. Anthocyanin concentrations were determined using a published acidified methanol method. Hot pepper fruits were first ground with liquid nitrogen. Acidified methanol was then mixed with the ground materials for 10 min in darkness with shaking. These crude extracts were subsequently mixed with an extraction solvent containing 1:1 chloroform:water to isolate anthocyanins. After centrifugation, the absorbance of the supernatant was read at 530 and 657 nm by the spectrophotometer, and anthocyanin contents were calculated from these values. The effects of pH were also examined by culturing strain 869T2 in LB media at 30 ◦C over a pH range of 4 to 9. Strain 869T2 was able to grow over this entire pH range . The results shown in Figure 1D demonstrate that IAA production was at a similar level when bacteria were grown at pH 6 to 9, whereas the IAA amount decreased 44.0% when bacteria were grown at pH 4. Additionally, three different sugars, glucose, fructose, and sucrose, were used in the minimal medium to examine the effects of different carbon sources on IAA production.
Strain 869T2 grew similarly in the M9 salt media with different kinds of sugars . The results shown in Figure 1F indicate that when strain 869T2 was grown in the media with two kinds of monosaccharide, glucose and fructose, the IAA amounts were higher than for the bacteria grown in the media with sucrose. We further investigated whether strain 869T2 had other plant-growth-promoting traits, including siderophore production and phosphate solubilization abilities, with agar plate assays. Supplementary Materials Figure S1A shows that the strain 869T2 colonies exposed to CAS agarose turned yellow, indicating the siderophore production ability of strain 869T2. Furthermore, Figure S1B reveals that the formation of halos around the strain 869T2 colonies grown in Pikovskaya’s agar medium with 0.5% tricalcium phosphate suggests that strain 869T2 may have the ability to solubilize phosphate.A previous study by Ho et al. demonstrated that strain 869T2 promoted plant growth in banana, a monocot. Here, the growth promotion ability of strain 869T2 was tested in three different eudicot plants from the Brassicaceae family, namely Arabidopsis thaliana, ching chiang pak choi, and pak choi. Because strain 869T2 produced relatively higher amounts of IAA at 25 ◦C to 37 ◦C , we cultured strain 869T2 at three different temperatures, 25 ◦C, 30 ◦C, and 37 ◦C. Subsequently Arabidopsis thaliana ecotype Columbia was inoculated with these strains to determine which strain had the best plant growth promotion ability. We confirmed the endophytic colonization of the Arabidopsis plants by strain 869T2 by reisolating the bacteria from surface-sterilized inoculated plant tissues. The identities of the isolated bacteria were determined via sequencing and phylogenetic analysis of the 16S ribosomal RNA gene. Subsequently, different plant growth parameters were examined in Arabidopsis plants inoculated with strain 869T2 and in mockinoculated controls. Two weeks after inoculation, the presence of strain 869T2 increased the average fresh weight , rosette diameter , root length , number of leaves , total leaf area per plant , leaf area per leaf , number of inflorescences , and number of siliques of Arabidopsis plants more than 1.5- to 2.1-fold compared with mock-inoculated controls. As shown in Figure 2I–K, the overall size and number of leaves of plants inoculated with strain 869T2 were greater than those of control plants, indicating that strain 869T2 promoted Arabidopsis plant growth. Furthermore, when the plants were inoculated with strain 869T2 grown at 30 ◦C, the average root length and average total leaf area per plant were slightly higher than for the strains grown at 25 ◦C and 37 ◦C.