Auxin production by plant associated bacteria: impact on endogenous IAA content and growth of Triticum aestivum L.

Authors


Shahida Hasnain, Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan. E-mail: genetic@brain.net.pk

Abstract

Aims:  The aim of this study was to investigate the potential of bacterial strains of Bacillus, Pseudomonas, Escherichia, Micrococcus and Staphylococcus genera associated with wild herbaceous flora to enhance endogenous indole-3-acetic acid (IAA) content and growth of Triticum aestivum var. Inqalab-91.

Methods and Results:  Gas chromatography and mass spectrometric (GC–MS) analysis revealed that bacterial strains produced 0·6–8·22 μg IAA ml−1 in the presence of L-tryptophan. Plant microbe experiments showed a significant positive correlation between auxin production by bacterial strains and endogenous IAA content of T. aestivum for GC–MS (r = 0·618; P = 0.05) and colorimetric analysis (r = 0·693; P = 0.01). Similarly, highly significant positive correlation for shoot length (r = 0·627; P = 0.01) and shoot fresh weight (r = 0·626; P = 0.01) was observed with auxin production under axenic conditions. Bacterial inoculations also enhanced shoot length (up to 29·16%), number of tillers (up to 97·35%), spike length (up to 25·20%) and seed weight (up to 13·70%) at final harvest.

Conclusions:  Bacterial strains have the ability to increase the endogenous IAA content and growth of T. aestivum var. Inqalab-91.

Significance and Impact of the Study:  Microbial strains of wild herbaceous flora can be effectively used to enhance the growth and yield of agronomically important crops.

Introduction

Indole-3-acetic acid (IAA) is an important phytohormone that coordinates different developmental processes in plants. IAA production is also wide spread among plant associated bacteria and play very critical role in plant growth and development (Costacurta and Vanderleyden 1995; Khalid et al. 2004). It is very likely that plant growth promotion by rhizobacteria is the result of combined action of several ways, but production of phytohormones (especially IAA) is considered as a direct mechanism used by bacteria to increase the growth and yield of plants (Arkhipova et al. 2005; Idris et al. 2007). The role of plant growth promoting bacteria (PGPB) have been extensively studied as biofertilizers to increase the yield of agronomically important crops such as wheat (Khalid et al. 2004), corn (Mehnaz and Lazarovits 2006) and lettuce (Arkhipova et al. 2005). Biosynthesis of IAA is considered very crucial in plant growth and development; therefore, the potential of microbial strains to enhance in planta IAA content can be used as a basic criterion for the selection of effective PGPB. The main objective of this research work was to evaluate the ability of bacterial strains associated with wild herbaceous flora to enhance in planta IAA content and growth of Triticum aestivum L. var. Inqalab-91 under axenic and wire house conditions. Bacterial strains from these plants are not reported for their growth promoting activities in relation to their auxin production.

Materials and methods

Bacterial strains and growth conditions

Bacterial strains used in this study that were isolated from the rhizosphere, histoplane and phyllosphere of wild herbaceous plants are listed in Table 1. Strains were routinely grown on L-broth or L-agar medium at 37°C (Gerhardt et al. 1994).

Table 1.   16S rDNA gene sequencing of bacterial strains associated with different plant species
StrainsPlantSourceIdentified asAccessions
ChR-1Chenopodium albumRhizosphereBacillus sp. ChR-1EU276629
FiR-1Fragaria indicaRhizosphereBacillus sp. FiR-1EU276630
NpR-1Nicotiana plumbaginifoliaRhizosphereBacillus sp. NpR-1EU276631
MiR-4Mellilotus indicaRhizosphereBacillus sp. MiR-4EU368168
AaH-1Anagalis arvensisHistoplaneBacillus sp. AaH-1EU368170
EhH-5Euphorbia helioscopiaHistoplaneBacillus sp. EhH-5EU368172
BP-1Boerhavia diffusaPhyllosphereBacillus sp. BP-1EU368179
EpP-2E. prostrataPhyllosphereBacillus sp. EpP-2EU368180
TpP-1Trianthema partulacastrumPhyllosphereBacillus sp. TpP-1EU368182
AvR-2Amaranthus viridisRhizospherePseudomonas sp. AvR-2EU190452
AvH-4A. viridisHistoplanePseudomonas sp. AvH-4EU190453
As-17Asphodalus tenuifoliusRhizospherePseudomonas sp. As-17EU368175
SnR-1Solanum nigrumRhizosphereEscherichia sp. SnR-1EU368169
AvR-5A. viridisRhizosphereMicrococcus sp. AvR-5EU368178
CdR-1Coronopus didymusRhizosphereStaphylococcus sp. CdR-1EU276627

16S rDNA sequencing

Genomic DNA was extracted from overnight-grown bacterial cultures incubated at 37°C in Luria-Bertani broth medium at 120 rev min−1 (Oxoid). DNA extraction was carried out by using AquaPure genomic DNA isolation kit (Bio-Rad) following the manufacturer’s instructions. Amplification of 16S rDNA was performed according to the method described by Hasnain and Thomas (1996). A total of 1·5-kb DNA fragment containing rRNA gene was amplified using forward primer 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1522r [5′-AAGGAGGTGATCCA(AG)CCGCA-3′] (Johnson 1994). The product was purified using QIAquick gel extraction kit (Qiagen) and sequenced using 27f primer by ABI PRISM-3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Colorimetric and GC–MS analysis of bacterial auxin production

Strains were grown in 100 ml L-broth medium in 250 ml Erlenmeyer flasks supplemented with 10 ml filter sterilized solution of L-tryptophan to a final concentration 1 mg ml−1. The flasks were inoculated with 100 μl of bacterial cell suspension adjusted to optical density of 0·5 (107 CFU ml−1) measured at 600 nm by spectrophotometer (S-300D; R & M Marketing, Hounslow, UK). All flasks were incubated at 37°C for 72 h at 120 rev min−1 in triplicate. After incubation, cells were removed from culture medium by centrifugation at 2300 g for 15 min (Sigma 2-5; Sigma Laborzentrifugen, Osterode, Germany) and auxin was detected in 1 ml of supernatant using Salkowski reagent as described earlier (Ali and Hasnain 2007). For gas chromatography and mass spectrometric (GC–MS) analysis, 10 ml of stationary phase cultures were extracted three times with equal volume of ethyl acetate, dried and re-dissolved in 2 ml HPLC grade methanol. About 20 μl methanolic extract was dissolved in 0·5 ml of 50 mmol l−1 sodium phosphate buffer (pH 7·0) containing 0·02% diethyldithiocarbamic acid (as an antioxidant) and 5 ng 13C6IAA was added as an internal standard. Bacterial samples were purified, derivatized and quantified by GC–MS as described by Andersen et al. (2008).

Measurements of endogenous IAA concentrations

Effect of bacterial inoculation on endogenous IAA content of T. aestivum under in vitro conditions was evaluated by growing plants in glass tubes (25 × 160 mm), each containing 25 g moistened autoclaved sand. Seeds procured from Punjab Seed Corp., Lahore, Pakistan, were surface sterilized by 0·1% solution of HgCl2 and washed 3–4 times in autoclaved distilled water. Two days old seedlings were inoculated with 500 μl of bacterial suspension adjusted to approximately 107 CFU ml−1 and 500 μl filter sterilized solution of L-tryptophan (1 mg ml−1) was also injected within rhizosphere, as precursor for IAA biosynthesis. In addition to water treated seeds, L-tryptophan amended control that also received 500 μl of precursor and L-broth was also included for comparison. Seedlings were incubated in Versatile Environmental Test Chamber (MRL-350H; Sanyo, Osaka, Japan) at 25 ± 1°C under photoperiod of 16 h with light intensity of 50–55 μmol s−2 m−1. Leaf tissue was collected after 14 days of seedling growth and immediately frozen in liquid nitrogen for quantification of free IAA content as described in Andersen et al. (2008).

Biological activity of bacterial auxin

Biological activity of bacterial IAA was demonstrated by evaluating the impact of stationary phase cultures on root growth of T. aestivum under axenic conditions. Petri plates with two filter papers were autoclaved and soaked with 10 ml sterilized distilled water. Five sterilized seeds were placed in each petri plate in duplicate and 2 ml stationary phase cultures grown in the presence of 1 mg ml−1 L-tryptophan in L-broth were poured. Water treated seeds were used as control and all Petri plates were incubated in Versatile Environmental Test Chamber as mentioned above.

Plant growth experiments

Pot experiments were conducted to evaluate the phytostimulatory effect of bacterial strains on T. aestivum under axenic and wire house conditions. Sterilized seeds of T. aestivum were incubated in bacterial suspension adjusted to optical density of 0·5 containing approximately107 CFU ml−1 for 15 min. Water treated seeds were used as control. All pots, plastic trays and covers used during the experiment were sterilized with 5% solution of sodium hypochlorite for 20 min. Seeds were inoculated in pot (6·5 × 6·5 cm) containing autoclaved soil mixture and vermiculite in 1 : 1 ratio under fully axenic conditions. Eight seeds were sown in each pot in triplicate and experiment was repeated twice. After germination, thinning was carried out to leave five seedlings in each pot and growth parameters in terms of shoot length and shoot fresh weight were analysed after 2 weeks growth period. Experiment was conducted at 22 ± 2°C temperature, 50% humidity and 12 h photoperiod with light intensity of 150–200 μmol m−2 s−1.

For wire house experiment seeds were processed as mentioned above and sown in larger pots (30 × 30 cm) containing 10 kg of unfertilized garden soil (pH 7·2; EC 44 ds m−1; organic content 0·60%). Initially, 18 seeds were inoculated in each pot in six replicates and after germination within 10–12 days seedlings were thinned to 15 per pot. All pots were arranged in completely randomized design. Experiment was conducted from December, 2006 to April, 2007 in the wire house of Department of Botany, University of the Punjab, Lahore, Pakistan, under ambient light and temperature. After 6 weeks of growth, further thinning was accomplished by keeping 10 seedlings per pot, which were grown till maturity. At full maturity, 10 plants were harvested from each pot to measure different growth parameters including shoot length, number of tillers, spike’s length and weight of 100 seeds.

Statistical analysis

For all experiments, the data were subjected to statistical analysis using software spss 12 program (SPSS Inc., Chicago, IL). Data were subjected to analysis of variance (anova) and means separated using Duncan’s multiple range test (P = 0·05). The correlation coefficients between auxin production and different growth parameters were also calculated.

Results

16S rDNA sequencing

Sequences of 16S rDNA were compared with sequence database (GenBank) through Blast (http://www.ncbi.nlm.nih.gov/BLAST). On the basis of homology, strains ChR-1, FiR-1, NpR-1, MiR-4, AaH-1, EhH-5, BP-1, EpP-2 and TpP-1 had maximum similarity of 98%, 99%, 100%, 100%, 99%, 100%, 100%, 100% and 100% with genus Bacillus respectively. Strains AvR-2, AvH-4 and As-17 showed 97%, 97% and 100% similarity with genus Pseudomonas and SnR-1, AvR-5 and CdR-1 had maximum homology of 97%, 100% and 99% with Escherichia, Micrococcus and Staphylococcus respectively. The nucleotide sequences obtained in this study had been submitted to GenBank under different accession numbers (Table 1).

Quantification of IAA

All bacterial strains showed the ability to synthesize IAA in the presence of precursor L-tryptophan. Strains varied in their potential of IAA production, and even strains belonging to same genera such as Bacillus and Pseudomonas produced different amounts of IAA in liquid culture medium. Auxin concentrations determined with Salkowski reagent ranges from 14 to 106 μg ml−1. The most active producers of IAA were Pseudomonas sp. As-17, Bacillus sp. MiR-4, Bacillus sp. TpP-1, Bacillus sp. NpR-1, Bacillus sp. BP-1, Bacillus sp. FiR-1 and Bacillus sp. ChR-1. GC–MS analysis of IAA revealed huge differences in culture medium as compared with colorimetric method (Table 2). Diagnostic ions for 13C6IAA and IAA were m/z 208 and m/z 202 respectively.

Table 2.   Colorimetric and gas chromatography and mass spectrometric (GC–MS) quantification of bacterial indole-3-acetic acid (IAA)
S. No.StrainsIAA concentration (μg ml−1)
ColorimetricGC–MS
  1. Mean of three replicates.

  2. Different letters within same column indicate significant difference between strains using Duncan’s multiple range test (P = 0·05).

 1Bacillus sp. ChR-148·3 bc0·94 a
 2Bacillus sp. FiR-154·2 bc0·70 a
 3Bacillus sp. NpR-164·0 c2·83 c
 4Bacillus sp. MiR-492·7 de3·00 d
 5Bacillus sp. AaH-114·3 a0·83 a
 6Bacillus sp. EhH-533·4 ab0·70 a
 7Bacillus sp. BP-154·3 bc0·77 a
 8Bacillus sp. EpP-214·4 a0·60 a
 9Bacillus sp. TpP-165·4 c0·72 a
10Pseudomonas sp. AvR-232·4 ab3·63 d
11Pseudomonas sp. AvH-472·3 cd7·04 f
12Pseudomonas sp. As-17106 e8·22 g
13Escherichia sp. SnR-119·1 a4·27 e
14Micrococcus sp. AvR-523·0 a1·16 a
15Staphylococcus sp. CdR-134·5 ab2·18 b

Endogenous IAA content of Triticum aestivum

Most of the bacterial strains of Pseudomonas and Bacillus genera enhanced IAA content of T. aestivum under in vitro conditions (Fig. 1). Pseudomonas strains were more effective to increase IAA content as compared with Bacillus. For instance, Pseudomonas sp. As-17 and Pseudomonas sp. AvH-4 increased endogenous IAA by 208% and 187% respectively. Bacillus strains MiR-4, NpR-1, AaH-1 and BP-1 increased endogenous IAA content 140%, 108%, 105% and 104% respectively, over water treated control. Significant positive correlation (r = 0·618; P = 0.05 and r = 0·693; P = 0.01) was observed between bacterial auxin production and endogenous IAA content analysed by GC–MS and colorimetric methods respectively.

Figure 1.

 Effect of bacterial inoculations on endogenous indole-3-acetic acid (IAA) content (ng mg−1 fresh weight) of Triticum aestivum. Abbreviations/affiliations: Trp, Tryptophan; SnR-1, Escherichia; AvR-5, Micrococcus; CdR-1, Staphylococcus. Mean of three replicates. Different letters indicate significant difference between treatments, using Duncan’s multiple range test (P = 0·05).

Growth experiments under axenic conditions

Bacterial inoculations caused an inhibition of primary root elongation followed by an increase in lateral root number, that might indicate the synthesis of IAA by bacteria in stationary phase cultures (Table 3). All bacterial treatments decreased the root length but enhanced the number of lateral roots and caused hypertrophy (in width-circumference), over control. The highest increase in number of roots over water treated control was 83·25% with Pseudomonas sp. As-17 and 75% with Bacillus sp. NpR-1, Bacillus sp. EhH-5 and Micrococcus sp. AvR-5.

Table 3.   Effect of auxin producing bacterial strains on growth of Triticum aestivum var. Inqalab-91 under axenic conditions
StrainsPot experimentsRoot growth experiments*
Shoot length (cm)Shoot fresh weight (g)Root length (cm)No. of roots
  1. Mean of two repeated experiments (30 plants).

  2. Different letters within same column indicate significant difference between treatments using Duncan’s multiple range test (P = 0·05).

  3. *Mean of two repeated experiments (10 seedlings).

Control19·10 abc2·00 a5·66 d4·00 a
ChR-121·10 cd2·00 a1·83 ab6·33 ab
FiR-119·80 abcd2·13 ab1·20 a4·00 a
NpR-121·26 d2·70 a1·66 ab7·00 b
MiR-420·13 bcd2·60 bcd4·00 c5·33 ab
AaH-118·13 a2·11 a4·13 c6·66 ab
EhH-519·26 abcd2·15 ab3·93 c7·00 b
BP-120·50 cd2·10 a1·80 ab4·66 ab
EpP-219·30 abcd2·70 cd2·10 ab6·66 ab
TpP-119·43 abcd2·15 ab1·76 ab6·66 ab
AvR-220·20 bcd2·25 abc1·00 a6·00 ab
AvH-420·96 cd2·80 d1·63 ab5·66 ab
As-1721·20 d2·70 cd3·86 c7·33 b
SnR-120·93 cd2·27 abc3·00 c5·00 ab
AvR-518·43 ab1·88 a1·33 a7·00 b
CdR-120·33 bcd2·74 cd1·76 ab6·00 ab

In pot experiments, most of the bacterial strains belonging to different genera increased vegetative growth parameters under axenic conditions. Maximum increase in shoot length and fresh weight was observed with Bacillus sp. NpR-1 (11·30%) and Pseudomonas sp. AvH-4 (40%) respectively over control (Table 3). Shoot length (r = 0·627; P = 0.01) and shoot fresh weight (r = 0·626; P = 0.01) recorded highly significant correlation with auxin production for GC–MS analysis. Also a significant correlation for shoot length (r = 0·585; P = 0.05) was found with auxin production quantified by colorimetric method.

Growth experiments under natural conditions

Growth responses in wire house under natural environmental conditions were also recorded after harvesting plants at full maturity (Table 4). The highest increases in shoot length over control were observed with Pseudomonas sp. AvH-4 (29·16%), Bacillus sp. EpP-2 (19·70%) and Bacillus sp. NpR-1 (19·56%). Bacterial inoculations significantly enhanced number of tillers especially with Bacillus sp. NpR-1, Pseudomonas sp. AvH-4, Bacillus sp. EpP-2 and Pseudomonas sp. AvR-2 with an increase of 97·35%, 82·74%, 70·80% and 68·10% respectively over control. Similarly, maximum increase in spike length and seed weight was 25·20% and 13·70% respectively with Bacillus sp. MiR-4. Significant correlation was found for shoot length (r = 0·575; P = 0.05) and seeds weight (r = 0·501; P = 0.05) at full maturity with auxin production revealed by GC–MS analysis.

Table 4.   Effect of bacterial inoculations on growth and yield of Triticum aestivum var. Inqalab-91 at full maturity under natural wire house conditions
StrainsShoot length (cm)No. of tillersSpike length (cm)Weight of 100 seeds (g)
  1. Mean of six replicates (60 plants).

  2. Different letters within same column indicate significant difference between treatments using Duncan’s multiple range test (P = 0·05).

Control52·80 a2·26 ab 7·86 a2·85 ab
ChR-159·37 cdef3·00 abcd8·70 abcde2·92 abc
FiR-159·73 bdef3·26 bcde8·23 ab3·00 abcd
NpR-163·13 f4·46 f7·94 a3·20 cd
MiR-461·40 ef2·73 abc9·84 e3·24 d
AaH-154·00 ab2·13 a8·62 abcde3·00 abcd
EhH-559·60 cdef3·00 abcd9·40 bcde3·20 cd
BP-155·93 abcd2·66 ab8·36 abc2·80 a
EpP-263·20 f3·86 def8·30 abc3·00 abcd
TpP-158·20 bcde2·33 ab8·76 abcde2·82 a
AvR-255·12 abc3·80 cdef9·75 de3·00 abcd
AvH-468·20 g4·13 ef8·46 abcd3·13 bcd
As-1762·13 ef2·46 ab9·62 cde3·13 bcd
SnR-162·86 f3·40 bcdef8·45 abcd3·23 d
AvR-560·53 ef3·40 bcdef8·72 abcde3·13 bcd
CdR-155·86 abcd3·00 abcd8·85 abcde2·94 abcd

Discussion

Present work demonstrated the phytostimulatory effect of bacteria associated with wild plant species under axenic and wire house conditions. Colorimetric and GC–MS analysis showed that bacteria differ in their ability to produce auxin in the presence of L-tryptophan. However, quantification by these two techniques indicated huge differences in IAA content. Salkowski reagent is widely used for the quantification of IAA from bacterial culture supernatant. But different intermediates of IAA biosynthetic pathways can also react with Salkowski reagent and contributes to colour absorption during quantification (Glickmann and Dessaux 1995). On the other hand, GC–MS analysis offer high sensitivity and the analysis can be performed with great accuracy and precision using 13C6IAA as internal standard (Ljung et al. 2004). The biological activity of bacterial IAA was demonstrated by its inhibitory effect on root length and increase in lateral root number of T. aestivum, indicating the production of IAA from L-tryptophan. Barazani and Friedman (2000) also reported that high concentration of L-tryptophan inhibited root elongation of lettuce seedlings because of excessive secretion of IAA. Although the role of IAA in root growth and development is well established, nitric oxide and N-acetyl homoserine lactone, a quorum sensing signals of bacteria have also been reported to mediate root growth independent of auxin signalling (Molina-Favero et al. 2008; Ortíz-Castro et al. 2008). Rhizobacteria can have marked effects on plant growth by contributing an exogenous source of phytohormones (Arkhipova et al. 2005; Cohen et al. 2008). In our experiment, positive correlation between bacterial auxin production and endogenous IAA content of T. aestivum was observed for GC–MS (r = 0·618; P = 0.05) and colorimetric analysis (r = 0·693; P = 0.01) that indicated the crucial role of bacterial auxin in plant growth. Idris et al. (2007) demonstrated the relationship of plant growth efficiency with IAA synthesizing ability of Bacillus amyloliquefaciens.

In the end, it can be concluded that bacterial strains isolated from wild herbaceous flora enhanced in planta IAA content and this trait can be used for the selection of effective PGPB. Pot experiments demonstrated that strains belonging to Bacillus, Pseudomonas, Staphylococcus and Escherichia have the potential to increase the growth and yield of T. aestivum. Strains offer a good opportunity to be used as biofertilizers to increase the growth and yield of agronomically important crops.

Acknowledgements

Higher Education Commission of Pakistan is acknowledged for providing funding to Basharat Ali (IRSIP No. 1-8/HEC/HRD/2007/471-VI) to visit Umeå Plant Science Centre, Sweden to do GC–MS quantifications of IAA. We thank Roger Granbom for help with GC–MS analysis.

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