Gibberellin Production by Newly Isolated Strain Leifsonia soli SE134 and Its Potential to Promote Plant Growth

Abstract

Very few plant growth-promoting rhizobacteria (PGPR) are known to produce gibberellins (GAs). The current study aimed to isolate a phytohormone-producing PGP rhizobacterium from soil and assess its potential to enhance plant growth. The newly isolated bacterium was identified as Leifsonia soli sp. SE134 on the basis of partial 16S ribosomal RNA gene sequence. Application of L. soli culture filtrate significantly increased the biomass, hypocotyl, and root lengths of cucumber seeds as compared with non-inoculated sole medium and distilled water treated controls. Furthermore, the PGPR culture was applied to the GA-deficient mutant rice cultivar Waito-C. Treatment with L. soli SE134 significantly increased the growth of Waito-C rice seedlings as compared with controls. Upon chromatographic analysis of L. soli culture, we isolated, detected and quantified different GAs; namely, $GA_1$ ($0.61{\pm}0.15$), $GA_4$ ($1.58{\pm}0.26$), $GA_7$ ($0.54{\pm}0.18$), $GA_8$ ($0.98{\pm}0.15$), $GA_9$ ($0.45{\pm}0.17$), $GA_{12}$ ($0.64{\pm}0.21$), $GA_{19}$ ($0.18{\pm}0.09$), $GA_{20}$ ($0.78{\pm}0.15$), $GA_{24}$ ($0.38{\pm}0.09$), $GA_{34}$ ($0.35{\pm}0.10$), and $GA_{53}$ ($0.17{\pm}0.05$). Plant growth promotion in cucumber, tomato, and young radish plants further evidenced the potential of this strain as a PGP bacterium. The results suggest that GA secretion by L. soli SE134 might prove advantageous for its ameliorative role in crop growth. These findings can be extended for improving the productivity of different crops under diverse environmental conditions.

Materials and Methods

Isolation of PGPR and Effect on Crop Seed Germination

Soil samples were collected from various agricultural fields in the Daegu area (Republic of Korea). About 10 g of soil samples was transferred to 250 ml flasks containing 100 ml sterile Amies solution [3]. The resulting suspensions were serially diluted (10-4) and 0.1 ml aliquots were grown on tryptic soy/agar (TSA; Merck Co., Germany) for isolation of rhizobacteria. Bacterial cultures were incubated for 48 h at 30℃, and bacterial colonies differentiated by their morphology, pigmentation, and growth rate were selected, counted, and restreaked on fresh TSA medium. For long-term preservation, bacteria were stored in 50% glycerol at -80℃.

In order to investigate the effect of bacterial culture on seed germination, cucumber seeds were purchased from Seminis Korea Co. (Korea), surface sterilized with NaOCl (5%) for 10 min, and thoroughly rinsed with distilled water (DW). Seeds were kept in a petri dish at 25 ± 2℃ in a culture room. The seeds were treated with ten dilutions of bacterial culture broth (30℃, 200 rpm and 3 days). DW and nutrient broth (NB) were used as controls for this experiment.

Bioassay of Gibberellin Mutant Waito-C Rice

Production of plant growth-promoting metabolites in PGPR was analyzed by performing a screening bioassay on gibberellin biosynthesis deficient mutant Waito-C rice. Waito-C rice seeds were surface sterilized with 2.5% sodium hypochlorite for 30 min, rinsed with autoclaved DW, and incubated for 24 h with 20 ppm uniconazol [19] to obtained equally germinated seeds. The germinated seeds were grown on agar medium (0.8%) in a growth chamber (day/night cycle: 14 h; 28℃/10 h; 18℃; relative humidity 60–70%; light intensity 1,000 µmm-2s natrium lamps) for 10 days. After the two-leaves stage was attained, 10 µl of culture filtrate solution of isolated bacteria was applied at the apex of Waito-C rice seedlings. One week after treatment, the shoot length, chlorophyll content, and shoot fresh and dry weights were recorded and compared with those of DW and NB controls. The experiment was repeated twice and each treatment had three replications comprising nine plants per replica.

Identification and Phylogenetic Analysis

The isolated bacterial strain was identified on the basis of its partial 16S ribosomal RNA gene (rDNA) sequence. The chromosomal DNA was isolated through standard procedures [35]. The almost complete 16S rDNAs were PCR amplified using primers 27F (5’-AGAGTTTGATC (AC) TGGCTCAG-3’) and 1492R (5’-CGG (CT) TACCTTGTTACGACTT-3’), which are complementary to the 5’ end and3’ end of the prokaryotic 16S rDNA, respectively. The amplification reaction was performed as previously described [1]. The BLAST search program (http://www.ncbi.nlm.nih.gov/BLAST/) was used to look for nucleotide sequence homology of this bacterial isolate. Closely related sequences were aligned by ClustalW using MEGA ver. 5.0 software, and the neighbor-joining tree was generated using the same software. Bootstrap replication (1000 replications) was used for statistical support for the nodes in the phylogenetic tree.

Plant Growth Promotion Capacity of Leifsonia soli

Seeds of cucumber, tomato, and young radish purchased from Seminis Korea Co. were surface sterilized with NaOCl (5%) for 10 min, rinsed with distilled water, and treated with 20 ppm uniconazole. Seeds were washed again and sown in plastic pots under controlled greenhouse conditions at 30 ± 2℃. Plant seedlings were treated with 5 ml of bacterial suspension 14 days after sowing, and the growth attributes of shoot length, plant fresh weight, and chlorophyll content, were recorded after 14 days of treatment. The bacterial culture suspension was incubated for 3 days at 30℃ on a shaking incubator at 200 rpm. DW and NB (5 ml) were used as controls for this experiment. The experiment was repeated twice, while the individual treatment was repeated three times containing nine plants per treatment.

Extraction and Quantification of Gibberellins

Bacterial gibberellins were extracted from culture filtrates after 3 days of incubation according to an established protocol [27]. Extracted GAs were subjected to reverse-phase C18-HPLC. The GAs were chromatographed on a 3.9 × 300m Bondapak C18 column (Waters Corp., USA) and eluted at 1.5 ml/min with the following gradient: 0 to 5 min, isocratic 28% methanol (MeOH) in 1% aqueous acetic acid; 5 to 35 min, linear gradient from 28% to 86% MeOH; 35 to 36 min, 86% to 100% MeOH; 36 to 40 min, isocratic 100% MeOH. Up to 48 fractions of 1.5 ml each were collected. The fractions were then prepared for injection into a gas chromatograph/mass spectrometer (GC/MS) with selective ion monitoring mode (SIM) (6890N network GC system, and 5973 network mass selective detector; Agilent Technologies, USA). For each GA type, 1 µl of sample was injected into a DB-1 capillary column with a 30 m × 0.25 mm i.d. and 0.25 µm film thickness (J & W Scientific Co., USA). The GC oven temperature was programmed for a 1 min hold at 60℃, then to rise at 15℃/min to 200℃ followed by 5℃/min to 285℃. Helium carrier gas was maintained at a head pressure of 30 kPa. The GC was directly interfaced to a mass selective detector with an interface and source temperature of 280℃, an ionizing voltage of 70 eV, and a dwell time of 100 msec. Full-scan mode (the first trial) and three major ions of the supplemented [2H2] GA internal standards (obtained from Prof. Lewis N. Mander, Australian National University, Canberra, Australia) and bacterial gibberellins were monitored simultaneously. The retention time was determined using hydrocarbon standards to calculate the KRI (Kovats Retention Index) value, and GA quantification was based on peak area ratios of non-deuterated (extracted) GAs to deuterated GAs.

Statistical Analysis

The data were analyzed for standard error using Sigma Plot Software (10.0) and Student’s t-test was used to identify significant values. The mean values were compared using Duncan’s multiple range test at p ≤ 0.05 (ANOVA SAS release 9.1; SAS, Cary, NC, USA).

 

Results

Isolation of PGPR and Effect on Crop Seed Germination

Among 891 bacterial strains isolated from agricultural fields, SE134 was selected on the basis of its role in improving seed germination of cucumber. An SE134 culture was applied to the cucumber seeds. The bacteriafree culture medium (NB) and DW were applied to the seeds of cucumber as a control. The results showed that SE134 application significantly increased seed germination as compared with controls (Table 1). Fresh biomass and hypocotyl and root lengths were significantly higher in SE134-treated cucumber as compared with control applications.

Table 1.DW is distilled water and NB is nutrient broth. For each set of treatments, the different letters indicate significant differences (p < 0.05) between PGPR and controls as evaluated by Duncan's multiple range test. ± refers to SD of the mean of nine plants per treatment.

Gibberellin Mutant Waito-C Rice Bioassay

Waito-C rice is dwarf mutant rice with a deficient GA biosynthesis pathway. The purpose of this assay was to determine whether the rhizobacteria secrete any growth regulating substances that can overcome the growth deficiency of Waito-C rice. The results show that when SE134 culture filtrate was applied, Waito-C rice growth was significantly higher than that of the control plants (Table 2). The growthpromoting effect of SE134 was more highly significant than that of the rest of the bacterial strains as well as the negative control (data not shown).

Table 2.DW is distilled water and NB is nutrient broth. SL = shoot length; SFW = shoot fresh weight; SDW = shoot dry weight; CC = chlorophyll. For each set of treatments, the different letters indicate significant differences (p < 0.05) between PGPR and controls as evaluated by Duncan's multiple range test. ± refers to SE of the mean of nine plants per treatment.

Identification of Isolated Bacterium

A BLASTn search of the 16S rDNA sequence of SE134 showed 100% similarity with Leifsonia soli (Fig. 1). In the phylogenetic analysis, the sequence of the isolate showed 99% sequence homology with L. soli, indicating it to be a strain of this species. The 16S rDNA sequence of this strain was submitted to GenBank and was given the accession number KC819804.

Fig. 1.Identification of bacterium L. soli SE134 by 16S rRNA gene-based phylogenetic analysis.

Plant Growth Promotion Capacity of Leifsonia soli SE134

The growth-promoting capacity of isolate L. soli SE134 was bioassayed on cucumber, tomato, and young radish plants. The control included both DW and NB treatments (Table 3). L. soli SE134 bacterial suspension significantly promoted the growth of all seedlings. Shoot length, plant fresh weight, and chlorophyll were significantly higher as compared with control treatments.

Table 3.DW is distilled water and NB is nutrient broth. For each set of treatments, the different letters indicate significant differences (p < 0.05) between PGPR and controls as evaluated by Duncan's multiple range test. ± refers to SD of the mean of nine plants per treatment.

Extraction and Quantification of Gibberellins

L. soli SE134 was grown for 3 days in NB culture medium (30℃; 200 rpm) and centrifuged (4℃; 6000 rpm) to obtain a clear supernatant (100 ml). The supernatant was extracted and chromatographed to detect GAs. GC/MS-SIM analysis revealed that various physiologically active and inactive GAs were present in different quantities (Fig. 2). Physiologically bioactive GAs included GA1 (0.61 ± 0.15 ng/100 ml), GA4 (1.58 ± 0.26 ng/100 ml), and GA7 (0.54 ± 0.18 ng/100 ml). Physiologically inactive GAs were GA8 (0.98 ± 0.15 ng/100ml), GA9 (0.45 ± 0.17 ng/100 ml), GA12 (0.64 ± 0.21 ng/100 ml), GA19 (0.78 ± 0.15 ng/100 ml), GA20 (0.18 ± 0.09 ng/100 ml), GA24 (0.38 ± 0.09 ng/100 ml), GA34 (0.35 ± 0.10 ng/100 ml), and GA53 (0.17 ± 0.05 ng/100 ml). The quantity of physiologically bioactive GA4 was significantly higher as compared with other GAs.

Fig. 2.Gibberellin production by L. soli SE134. The bacterial culture was centrifuged and 100 ml of culture filtrate was analyzed for the presence of GAs using a GA extraction protocol [27].

 

Discussion

The use of PGPR has a potential role in the development of sustainable systems in crop production [37,38]. It not only helps to improve crop plant growth and yield but also avoids various forms of agricultural pollution [40]. Different strains of Alcaligenes, Arthrobacter, Azospirillium, Azotobacter, Beijerinckia, Bacillus, Enterobacter, Burkholderia, Acinetobacter, Erwinia, Flavobacterium, Rhizobium, and Serratia [33,34,39] have been identified as PGPR. These studies have suggested using PGPR as biofertilizers [2,28]. Crop plants are protected by PGPR that may produce secondary metabolites, such as alkaloids, antibiotics, and toxins [9,36]. Very little work has been performed on the synthesis and secretion of plant growth regulators from bacteria. In the present study, we assayed L. soli SE134 culture on cucumber and Waito-C rice seedlings. Among tested strains, L. soli SE134 culture filtrate significantly promoted the growth of cucumber and Waito-C rice seedlings, suggesting the presence of active plant growth metabolites. Waito-C is a dwarf and bioactive GA-deficient rice cultivar, with a blocked C13-hydroxylation pathway for GA biosynthesis [19]. To observe the sole effect of L. soli SE134 as a GA producer, Waito-C seeds were treated with uniconazol to further suppress endogenous GA production by blocking its biosynthesis pathway [19]. Using rice seedlings to elucidate the effect in a controlled environment is beneficial and easy to monitor, while using Waito-C rice can help to analyze the effect of GA-producing strains.

Gibberellin production by PGPR promotes the growth and yield of many crop plants. The plant’s endogenous gibberellin-glucosyl conjugates are released via root exudation by a process of deconjugation [32], while bacterial enzymes 3β-hydroxylize the inactive 3-deoxy gibberellins to active forms such as GA1, GA3, and GA4 [6,7,31]. The first report of gibberellin characterization in bacteria using physicochemical methods was by Atzorn et al. [4], who demonstrated the presence of GA1, GA4, GA9, and GA20 in Rhizobium meliloti culture solution. Similarly, the current study confirms the previous findings of Joo et al. [22] on 3β-hydroxylated GAs in B. cereus MJ-1, B. macroides CJ-29, and B. pumilus CJ- 69. The authors also showed the usefulness of HPLC coupled with GC-MS-SIM while suggesting an efficient way to isolate, detect, and quantify GA.

Bastian et al. [5] detected the phytohormones GA1 and GA3 in chemically defined cultures of Acetobacter diazotrophicus and Herbaspirillum seropedicae. Both bacteria are associated with Gramineae species in the endophytic mode of life and were found to promote plant growth and yield. Gutierrez-Manero et al. [14] isolated the PGPR Bacillus pumilus and Bacillus licheniformis from the rhizosphere of alder (Alnus glutinosa [L.]). Full-scan gas chromatography-mass spectrometry analyses on extracts of culture media showed the presence of GA1, GA3, GA4, and GA20 in addition to the isomers 3-epi-GA1 and iso-GA3.

Another PGP rhizobacterium, Acinetobacter calcoaceticus SE370, was found to be a novel GA producer, as it secretes 10 different GAs in its growth media [25], including physiologically active GA1, GA3, and GA4 [25]. The bioactive GA1, GA3, and GA4 content was 0.45, 6.25, and 2.83 ng per 100 ml, respectively. Similarly, another PGP strain from the rhizosphere, Promicromonospora sp. SE188, was found to produce physiologically active GA1 (0.99 ± 0.03 ng/ml) and GA4 (1.58 ± 0.11 ng/ml) and inactive GA9 (0.2 ± 0.04 ng/ml), GA12 (2.38 ± 0.11 ng/ml), GA19 (0.85 ± 0.07 ng/ml), GA20 (0.17 ± 0.08 ng/ml), GA24 (0.57 ± 0.07 ng/ml), GA34 (0.24 ± 0.03 ng/ml), and GA53 (0.29 ± 0.02 ng/ml) [26]. The GAs were analyzed through GC/MS in selected ion monitoring (SIM) mode, which provides a more reliable GA quantification technique as compared with TLC, bioassays, or HPLC-UV, which give poor resolution and the least degree of reliability. The major advantage of GC/MS is its unbiased character. In comparison with non-MS detection-based chromatographic techniques (HPLCDAD,GC-FID), where only compounds targeted by a special analytical protocol are found, GC-MS analysis can result in interesting and unexpected new information about a particular extract [11].

In the current study, bacterial culture suspensions significantly promoted the growth of cucumber, tomato, and young radish, which may be due to the GA production capacity of newly isolated L. soli SE134. However, this isolate had not been previously reported to produce gibberellins. L. soli SE134 was found to produce eleven GAs, including bioactive GA1, GA4, and GA7. Although the quantities of the GAs were lower than in Promicromonospora sp. SE188 and A. calcoaceticus SE370, GA secretion was higher in L. soli SE134. Additionally, we did not detect GA3 in L. soli SE134 culture, which suggests a different pathway of GA biosynthesis as compared with that of Bacillus pumilus, Bacillus licheniformis, Acetobacter diazotrophicus, Herbaspirillum seropedicae, and A. calcoaceticus SE370. However, the GA profile of L. soli SE134 is similar to that of Promicromonospora sp. SE188 and Rhizobium meliloti. The genus Leifsonia is classified in Actinobacteria [10], which contains twelve species and two subspecies, with L. aquatica as the type species [10,30]. Members of the genus Leifsonia have also been isolated from roots, soil, Himalayan glaciers, cyanobacterial mats, and water samples [8,10,15]. The present study is the first to report that a species of Leifsonia can also produce GAs.

In conclusion, the GA-secreting bacterium L. soli SE134 might be advantageous for plant growth promotion and metabolism. Its positive role in this regard was demonstrated by the measurement of plant growth attributes in cucumber and Waito-C rice. Understanding the interactions between PGPR and plants can improve the quality and quantity of crops. Such studies can be extended for improving agricultural productivity under the appropriate extreme environmental conditions.