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. 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, vertical farming systems and color of fruits of hot pepper and okra were recorded following previously described methods.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. We therefore tested whether strain 869T2 grown at 30 ◦C could enhance the growth of different Arabidopsis ecotypes and other plant species. In addition to Arabidopsis ecotype Columbia, three Arabidopsis ecotypes that are less susceptible to Agrobacterium tumefaciens infection, BL-1, UE-1, and Dijon-G, were selected to examine the growth promotion ability of strain 869T2. After inoculation with strain 869T2, the average value of the fresh weight , dry weight , rosette diameter , root length , number of leaves , total leaf area per plant , and leaf area per leaf of the three additional Arabidopsis ecotypes were 1.2- to 2.0-fold higher than control plants. These data further support the hypothesis that the presence of strain 869T2 in different Arabidopsis ecotypes has a positive impact on plant growth.Seedlings of ching chiang pak choi and pak choi from the Brassica genus were also inoculated with strain 869T2 to examine its effects on plant growth.

At 27, 33, and 40 days after inoculation with strain 869T2, the average fresh weight and dry weight of aboveground leaves of ching chiang pak choi were higher than those of the control plants. Furthermore, the average leaf length and width,vertical growing systems petiole length and width, number of leaves per plant, total leaf area per plant, and leaf area per leaf were greater in the 869T2-inoculated ching chiang pak choi compared to the control plants. The results shown in Figure 3J,K demonstrate that the average plant height and width of the 869T2-inoculated ching chiang pak choi were also greater compared to the control plants. Similarly, after the ching chiang pak choi was inoculated with strain 869T2, the average values of root fresh weight, dry weight, and length were higher in comparison to control plants. Figure 3O–Q indicate that both the aerial and below ground parts of ching chiang pak choi were larger after inoculation with strain 869T2. Figure 3R also shows that the ching chiang pak choi inoculated with strain 869T2 grew faster and flowered earlier than control plants 53 days after inoculation. Similarly, after inoculation with strain 869T2, the pak choi grew larger, including larger and more numerous leaves, larger aerial parts overall, and longer and heavier roots. These data indicate that inoculation of strain 869T2 in two vegetables from the Brassicaceae family significantly improved their growth. Because B. seminalis strain 869T2 successfully colonized Arabidopsis and two types of plants from the Brassicaceae family and promoted their growth, we further examined whether strain 869T2 could promote the growth of plants from the Asteraceae and Amaranthaceae families. At 35, 43, 50, and 56 days after inoculation with strain 869T2, the fresh weight of the aerial parts of inoculated loose-leaf lettuce plants increased 12.7- to 46.6-fold compared to the 0-day post-inoculation plants. By comparison, in the mock-inoculated control plants, the fresh weight increased 8.0- to 36.0-fold over the same period. Similarly, the dry weight of the inoculated loose-leaf lettuce increased more than that of the control plants at 35, 43, 50, and 56 days after inoculation. These data indicate that inoculation of the loose-leaf lettuce with strain 869T2 significantly enhanced plant growth. The weight increases of the inoculated loose-leaf lettuce plants were due to increases in average leaf width and length , the number of leaves per plant , total leaf area per plant and per leaf , and plant height and width. Furthermore, the root fresh weight of the inoculated loose-leaf lettuce plants increased 4.5- to 12.4-fold at 35, 43, 50, and 56 days after inoculation compared with the 0-day post-inoculation plants ; in contrast, that of the mock-inoculated control only increased 2.5- to 8.5-fold compared with the 0-day post-inoculation plants. 

Additionally, the root dry weight and length increased more in the inoculated loose-leaf lettuce plants than in the control plants. As seen in Figure 4M–O, overall plant size and leaf size increased after inoculation with strain 869T2, suggesting that strain 869T2 improves loose-leaf lettuce growth. We also inoculated strain 869T2 into romaine lettuce and red leaf lettuce. The results shown in Figures S4 and S5 demonstrate that both kinds of lettuce grew taller and wider, had more and larger leaves, and had heavier aerial and below ground tissues after inoculation with strain 869T2 compared with the control plants. The chlorophyll contents of red leaf lettuce leaves were also higher in the 869T2-inoculated plants than the control plants. These data collectively indicate that the three evaluated kinds of lettuce can grow significantly better after inoculation with strain 869T2. We also selected Chinese amaranth of the Amaranthaceae family to test the effect of strain 869T2 on its growth. At 36, 43, and 50 days after inoculation, the fresh weight of the 869T2-inoculated Chinese amaranth exhibited a 20.0- to 56.6-fold increase when compared to the 0-day post-inoculation plants, whereas the control plants only showed an 8.3- to 33.5-fold increase when compared to the 0-day post-inoculation plants. Other plant growth parameters of the 869T2-inoculated and control plants were also examined 36, 43, and 50 days after inoculation. Figure 5 illustrates that the 869T2-inoculated Chinese amaranth individuals had more and larger leaves, were taller and wider, and had heavier and longer roots than the control plants. These data show that inoculating strain 869T2 into Chinese amaranth promoted its growth.Because B. seminalis strain 869T2 promoted the growth of several leafy vegetables, we next tested the effects of the strain 869T2 on the flowering and fruit production of hot pepper and okra. Hot pepper plants, from the Solanaceae family, were inoculated with strain 869T2 but we did not observe significant growth promotion effects on the aerial and root parts of the plants. However, we did observe that the 869T2-inoculated hot pepper plants flowered 20 days after inoculation; the number of flowers continually increased and had more than a 7-fold increase at 37 days after inoculation. In the mock-inoculated control plants, we observed flowering 21 days after inoculation, and the number of flowers had only increased 5-fold at 37 days after inoculation. The average number of fruits on the 869T2-inoculated plants was higher than that on the control plants at 30, 37, 44, and 51 days after inoculation. The average numbers of flower buds, flowers, and fruits per plant were higher in the 869T2-inoculated plants than in the control plants beginning 21 days post-inoculation. Furthermore, the percentages of hot pepper fruits with red and green/yellow coloring were higher in the 869T2-inoculated plants than in the control plants 59, 66, 73, and 80 days after inoculation. Similarly, the average anthocyanin contents of the 869T2-inoculated plants were significantly higher than those of the control plants at 66, 73, and 80 days after inoculation. However, the average length, width, and fresh weight of the fruits were not significantly different between the inoculated and control plants. Collectively, these data suggest that the inoculation of hot pepper with strain 869T2 could increase flowering and fruiting in hot pepper plants and accelerate fruit maturation. We subsequently examined the effects of strain 869T2 on okra, which belongs to the Malvaceae family. The overall plant size and weight were not significantly different between the 869T2-inoculated and control okra plants. We observed, however, that the number of nodes of the first flower was smaller in the 869T2-inoculated okra than in the control plants, suggesting that the 869T2-inoculated okra plants flowered earlier than the control plants.