Microassay validation for bacterial IAA estimation as a new fine-tuned PGPR screening assay

Abstract

The detection and quantification of Indole -3 Acetic Acid (IAA) produced by Plant growth promoting rhizobacteria (PGPR) rely on a standard well-documented assay, which remains time-consuming, laborious, and costly. These drawbacks led to sway interest to economic and reliable assays. The aim of this work is to validate and standardize a fast, reliable, and cost-effective microassay to quantify IAA produced by bacteria with an easy microplate method. In order to validate the accuracy of the IAA microplate assay, bacterial samples from different genera were assayed using two methods: the conventional IAA estimation assay and the IAA micro- assay. The microassay shows a prominent reduction in used bacterial supernatant volume as well as Salkowski reagent volume of about 92.5%. It is considerably cheaper than the conventional one of around 56%. The newly performed microplate assay is 23 times faster. The result of IAA quantitative analysis for 13 bacterial strains showed that Bacillus muralis and Bacillus toyonensis produced the highest IAA concentration (23.64±0.003μg/ml and 23.35±0.006μg/ml, respectively). The obtained data from both methods were highly correlated with an R-value of 0.979. The microassay offers the ability to read the optical density of all samples simultaneously since used volumes of bacterial supernatants and Salkowski reagent were minimized to place the mixture in 96-well microplates, which reduces greatly required labor. Furthermore, the application of the IAA micro-plate assay reduces drastically the reagent waste and toxicity hazard of Salkowski reagent in the environment, thus, we can classify it as eco-friendly respecting the Green Chemistry concept according to Environmental Protection Agency (EPA). The IAA microassay is a, reliable, rapid and cost-effective and eco-friendly method to screen plant growth promoting potential of more than 23 bacterial strains by microplate. It could be an alternative for the conventional IAA assay as a routine research tool.

Keywords

PGPR Indole-3-acetic acid (IAA)microassay validation fast and cost effective eco-friendly

1 Introduction

Plants regulate their growth and development through small signaling molecules, named phytohormones. They play a crucial role in controlling growth, differentiation and multicellular communication in different plant parts [1]. These molecules circulate in all plant tissues to regulate the growth speed of individual parts and integrate these parts to produce the form that we recognize as a plant [2].

Phytohormones occur in plant as a group of low molecular weight molecules acting like stimulators such as auxins, gibberellins, cytokinin as well as natural inhibitors like abscisic acid, ethylene and phenolic inhibitors [3]. They act to mediate host responses to various biotic and abiotic stresses such as pathogen challenge, insect herbivory, drought, cold and heat stress [4, 5]. Figure 1 details the most important plant hormones and their impact on plant development.

Fig. 1

Role of phytohormones in regulating plant development.

Among various phytohormones, auxin represent the most important class regulating most of plant process [6]. It is involved in almost every aspect of plant development [7], from embryo state, to developing fruit [8, 9].

Auxin synthesis takes place in the stem ‘s tips, in the meristems and young leaves of the terminal buds. It is involved in several mechanisms like cell enlargement, cell division (in combination with cytokinin), vascular tissue differentiation, apical dominance, leaf and fruit abscission (using ethylene) [2] as well as root initiation and development [10].

Plants naturally face a complex microbial community that colonizes all plant parts including roots, stems and leaves [11]. Plant Growth Promoting Rhizobacteria (PGPR) are a group of bacteria that colonizes the rhizosphere and enhances plant growth and yield via various mechanisms [12].

Diverse bacterial species produce auxins as part of their metabolism. It includes indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or their precursors [13]. Tryptophan, an aromatic amino acid, found in abundance in root exudates, has been identified as the most important precursor for the biosynthesis of IAA in bacteria [14]. Two main pathways are involved in the biosynthesis of IAA, one uses tryptophan as a precursor, and the other is independent to tryptophan [15, 16]. Microbial biosynthesis of IAA is mostly based on tryptophan dependent pathway and it can occur through four different under pathways named (according to intermediate compounds) indole-3-acetamide, indole-3-pyruvic acid, indole-3-acetonitrile and tryptamine [17]. The indole-3-pyruvic acid (IPyA) pathway represents the main route of IAA biosynthesis in plants, plant pathogens and plant growth-promoting bacteria such as Pseudomonas, Azospirillum, Bacillus, Bradyrhizobium and Enterobacter among others [18, 19]. Figure 2 resumed the metabolic pathways of bacterial biosynthesis of IAA from L-tryptophan [20]. Bacterially produced IAA, when administered on plants, can affect the sensitivity of plant tissue, thus stimulating the plant growth, and having a positive impact on improving yields of crop productivity.

Fig. 2

Metabolic pathways of bacterial biosynthesis of IAA from L-tryptophan [20]. L-tryptophan is an aromatic amino acid which represents the initial precursor of Indole-3-acetic (IAA) acid biosynthesis in many Plant Growth Promoting Rhizobacteria (PGPR). These bacteria produce IAA according to their metabolic process. (A) Bacteria such as Agrobacterium and Rhizobium produces IAA via indole-3-acetamide pathway. (B) Other bacteria like Pseudomonas and Azospirillum synthesize IAA via Indole-3-pyruvic acid pathway to produce Indole-3-acetaldehyde which is converted to IAA (C) The genus Bacillus synthetize IAA via tryptamine pathway which convert Indole-3-ethanol to IAA.

In order to detect and quantify IAA in different biological matrices such as plants, algae, bacteria and fungi, several studies have described a number of possible methods, including analytical techniques namely gas chromatography (GC), capillary electrophoresis (CE) and liquid chromatography (LC) combined with several types of detectors like ultraviolet (UV), fluorescence (FL) or mass spectrometer (MS) [21]. Apart from the analytic methods, thin layer chromatography (TLC) method was also applied to estimate IAA produced by bacteria using silica gel plates. Produced IAA is identified under UV light by spraying the plates with Ehmann’s reagent [22]. Other reports were rather interested to estimate IAA produced by bacteria, namely PGPR, using qualitative method as well as spectrophotometric based method described by Gordon and Weber in 1951 [23, 24]. This conventional assay requires hard labor and needs a large number of test tubes and spectrophotometer cuvettes, in addition to the preparation of important reagent volume for many bacterial strains. These drawbacks led to sway interest to an economic and non-time-consuming quantification assays. The aim of this work is to validate and standardize a fast, reliable and cost effective microassay previously described by Sarwar and Kremer in 1995 [25] to quantify IAA produced by bacteria.

2 Materials and methods

2.1 Bacteria and growth conditions

In order to estimate IAA production by bacteria, 13 bacterial strains affiliated to different phyla: proteobacteria, firmicute and actinobacteria were selected from our laboratory collection (BVBGR, ISBST, Manouba University). Bacterial strains were grown in appropriate growth media supplemented with 0.5 g/L of L-Tryptophan. Cultures were incubated at 30°C for 24–48 h until we obtain an OD corresponding to 1±0.2 at 600 nm. All strains characteristics are detailed in Table 1.

Table 1
Characteristics of bacterial strains producing IAA

No Code Bacterial strain Gram Origin of isolates Phylogenetic group Culture medium Incubation temperature Accession number

1 B13 Buttiauxella brennerae Negative – Gamma proteobacteria TSB 30°C –

2 WH Pseudomonas brassicacearum Negative – Gamma proteobacteria TSB 30°C –

3 FR1 11 Bacillus megaterium Positive Fig rhizosphere Firmicute TSB 30°C MK590362

4 O3RR24 Pseudomonas brassicacearum Negative Olive rhizosphere Gamma proteobacteria YEM 30°C MK590373

5 O3RR17 Arthrobacter siccitolerans Positive Olive rhizosphere Actinobacteria YEM 30°C MK590372

6 FR1 17 Microbacterium azadirachtae Positive Fig rhizosphere Actinobacteria TSB 30°C MK590363

7 FR1 38 Brevibacterium frigotolerans Positive Fig rhizosphere Actinobacteria TSB 30°C MK590366

8 O3R52 Pseudomonas reinekei Negative Olive rhizosphere Gamma proteobacteria TSB 30°C MK590371

9 FR1 24 Bacillus toyonensis Positive Fig rhizosphere Firmicute TSB 30°C MK590364

10 O3RR35 Bacillus zhangzhouensis Positive Olive rhizosphere Firmicute YEM 30°C MK590370

11 O3R24 Bacillus muralis Positive Olive rhizosphere Firmicute TSB 30°C MK590374

12 O3RR33 Arthrobacter humicola Positive Olive rhizosphere Actinobacteria YEM 30°C MK590369

13 FR1 35 Bacillus wiedmannii Positive Fig rhizosphere Firmicute TSB 30°C MK590365

No	Code	Bacterial strain	Gram	Origin of isolates	Phylogenetic group	Culture medium	Incubation temperature	Accession number
1	B13	Buttiauxella brennerae	Negative	–	Gamma proteobacteria	TSB	30°C	–
2	WH	Pseudomonas brassicacearum	Negative	–	Gamma proteobacteria	TSB	30°C	–
3	FR1 11	Bacillus megaterium	Positive	Fig rhizosphere	Firmicute	TSB	30°C	MK590362
4	O3RR24	Pseudomonas brassicacearum	Negative	Olive rhizosphere	Gamma proteobacteria	YEM	30°C	MK590373
5	O3RR17	Arthrobacter siccitolerans	Positive	Olive rhizosphere	Actinobacteria	YEM	30°C	MK590372
6	FR1 17	Microbacterium azadirachtae	Positive	Fig rhizosphere	Actinobacteria	TSB	30°C	MK590363
7	FR1 38	Brevibacterium frigotolerans	Positive	Fig rhizosphere	Actinobacteria	TSB	30°C	MK590366
8	O3R52	Pseudomonas reinekei	Negative	Olive rhizosphere	Gamma proteobacteria	TSB	30°C	MK590371
9	FR1 24	Bacillus toyonensis	Positive	Fig rhizosphere	Firmicute	TSB	30°C	MK590364
10	O3RR35	Bacillus zhangzhouensis	Positive	Olive rhizosphere	Firmicute	YEM	30°C	MK590370
11	O3R24	Bacillus muralis	Positive	Olive rhizosphere	Firmicute	TSB	30°C	MK590374
12	O3RR33	Arthrobacter humicola	Positive	Olive rhizosphere	Actinobacteria	YEM	30°C	MK590369
13	FR1 35	Bacillus wiedmannii	Positive	Fig rhizosphere	Firmicute	TSB	30°C	MK590365

Each culture was centrifuged at 6000 RPM for 15 min in 4°C (EBA 200 –Hettich). Cell pellets were discarded and supernatants were taken carefully and stored at –20°C to estimate their IAA production later.

2.2 IAA estimation assay

Bacterial strains were assayed for their IAA-producing ability using Salkowski reagent, both qualitative and quantitative assays were performed. For both methods, Salkowski reagent is prepared by mixing 1.2 g FeCl₃ in 10 ml ultrapure water in a separate tube. Then, in an opaque glass flask, add 47.3 ml of pure H₂SO₄ and 42.7 ml ultrapure water. Finally add the FeCl₃ solution and mix carefully. The reagent should be kept in a tightly closed dark flask to avoid hazard of sulfuric acid [26].

2.2.1 Qualitative assay

The revelation of IAA production by bacterial strains was based on the shift of solution color to red-pinkish color. The intensity of the appeared color is proportional to IAA concentration.

2.2.2 Quantitative assay

Quantitative estimation of IAA production by bacterial strains was performed with conventional assay as well as the microassay in the same conditions of medium, temperature and concentrations.

2.2.3 Conventional IAA assay

We adopted the method of Gordon and Weber (1951) [23] with slight modifications. Briefly, 1 ml of bacterial supernatant was mixed with 2 ml of Salkowski reagent and incubated for 30 min in obscurity, at room temperature. A tube containing non inoculated broth and Salkowski reagent was maintained as a control. Optical density (OD) was determined in triplicate at 535 nm by a (Libra S22 - spectrophotometer UV-visible –BIOCHROM).

IAA concentration of each sample was determined from a calibration range of IAA carried out under the same conditions as all samples.

2.2.4 IAA microassay

The micro-assay was carried out in a 96 well microplate greiner bio-one CELLSTAR^® and the OD was carried out by a microplate reader (Thermo Scientific™ Multiskan™ FC -MIB 51119000). Briefly, 75μl of different bacterial supernatants were pipetted in separate wells of microplate, followed by the addition of 150μl of Salkowski reagent. After incubation of the microplate in a dark place, at room temperature for 25 min, the microplate was shacked and the optical density was taken at 535 nm with the microplate reader. Three replicates were performed for each strain as described in Fig. 3. IAA concentration was estimated based on IAA calibration range which was performed in the same microplate with the same experimental conditions.

Fig. 3

Protocol steps for IAA quantification with the microplate assay. 1: In a 96 well microplate, distribute different concentrations of IAA Standard solution horizontally in triplicate then, add 75μL of each bacterial supernatant in triplicate vertically in the rest of wells. Add 150μL of Salkowski reagent to all microplate wells and incubate the microplate for 25 min at room temperature in the microplate reader. 2: Shake the microplate for 30s and read the optical density with the microplate reader at 535 nm. 3: Obtained optical density data were resumed in a table identically divided as the microplate wells.

2.3 Statistical analysis: Correlation analysis between conventional method and newly performed microassay

In order to validate the correlation among conventional IAA estimation assay and the microplate assay, correlation coefficient, between different data obtained from both assays, was checked by R 3.5.1 free software.

3 Results and discussion

3.1 Bacterial strains

Thirteen bacterial strains were assayed for their ability to produce IAA. These strains, taken in the present study, belong to species amongst diverse genera including Pseudomonas, Buttiauxella, Bacillus, Brevibacterium, Arthrobacter and Micobacterium. Details of the chosen strains, such as collection site and culture conditions, were indicated in Table 1. Most of these strains are well-known PGPR. In fact, more than 80%of soil bacteria in the rhizosphere are capable to produce auxins [27, 28]. Indeed, Kisiel and Kępczyńska reported in 2016 that Pseudomonas brassicacearum increased the level of endogenous IAA in the leaves of M. truncatula.

3.2 IAA estimation assay

3.2.1 Qualitative estimation

IAA production is revealed by the appearance of red to pinkish color in the solution and this color intensity is proportional to IAA concentration. In fact, It was reported that indole acetic acid forms a chelate with iron at acidic pH [30]. The formed complex is called: Tris-(indole-3-acetato) iron III [31]. The oxidation of indole acetic acid by ferric salts is the basis of the colorimetric determination of the Salkowski reaction [23]. Proposed chemical reaction is showed in Fig. 4. It was clear that all bacterial strains, tested in this study, produced different IAA amounts as different shade of color were observed. Bacillus muralis (O3R24) and Bacillus toyonensis (Fr1 24) showed the most intense pinkish color in comparison to other strains. This method gives rough idea about IAA production. Hence, we resorted to quantifying IAA concentration in each bacterial supernatant by the conventional as well as the microplate assay.

Fig. 4

Chemical reaction of IAA quantification with Salkowski reagent.

3.2.2 Quantitative IAA estimation

The amount of IAA produced by the 13 bacterial strains was investigated and compared for both conventional and microplate assays in order to validate the accuracy of the microassay Table 2. Data in this Table show that Bacillus muralis (O3R24) produces the highest IAA concentration of about 23.64±0.003μg/ml. According to Yadav and his collaborators, this bacterial strain is considered as potent candidate to be developed as inoculants (with other bacterial genera) as it exhibited multiple PGP traits at low temperature [32]. Furthermore, B. muralis is identified as a potent IAA productive strain as it increased soil IAA concentration by 135.3%when inoculated [33].Obtained results indicate that Bacillus toyonensis (Fr1.24) is also a potent IAA productive strain with 23.35±0.006μg/ml. It was reported by Sen and his collaborators that B. toyonensis isolated from two ferns promotes plant growth by producing IAA as well as increasing nutrient uptake etc. [34]. A detailed study was conducted by Choudhury in 2017 demonstrating that B. toyonensis could produce up to 127.56μg/ml of IAA under optimal growth conditions [35].

Table 2
IAA production by bacterial strains estimated by conventional and performed microplate assays

No Bacterial strains Qualitative analysis Quantitative analysis

Conventional assay(μg/ml) Microplate assay(μg/ml)

1 B13 + 9.09±0.008 10.17±0.007

2 WH + 11.24±0.012 12.89±0.011

3 FR1 11 ++ 13.210±0.001 15.334±0.007

4 O3RR26 +++ 19.053±0.002 21.510±0.004

5 O3RR17 ++ 14.856±0.002 16.157±0.017

6 FR1 17 ++ 15.844±0.005 17.742±0.006

7 FR1 38 ++ 14.609±0.003 16.768±0.010

8 O3R52 ++ 14.239±0.002 13.934±0.007

9 FR1 24 +++ 20.905±0.006 23.359±0.006

10 O3RR35 ++ 12.140±0.056 13.460±0.005

11 O3R24 +++ 20.646±0.002 23.641±0.003

12 O3RR33 ++ 14.074±0.001 14.074±0.006

13 FR1 35 + 10.288±0.006 11.157±0.005

No	Bacterial strains	Qualitative analysis	Quantitative analysis
1	B13	+	9.09±0.008	10.17±0.007
2	WH	+	11.24±0.012	12.89±0.011
3	FR1 11	++	13.210±0.001	15.334±0.007
4	O3RR26	+++	19.053±0.002	21.510±0.004
5	O3RR17	++	14.856±0.002	16.157±0.017
6	FR1 17	++	15.844±0.005	17.742±0.006
7	FR1 38	++	14.609±0.003	16.768±0.010
8	O3R52	++	14.239±0.002	13.934±0.007
9	FR1 24	+++	20.905±0.006	23.359±0.006
10	O3RR35	++	12.140±0.056	13.460±0.005
11	O3R24	+++	20.646±0.002	23.641±0.003
12	O3RR33	++	14.074±0.001	14.074±0.006
13	FR1 35	+	10.288±0.006	11.157±0.005

Data are represented by the mean of three replicates±standard deviation; (+++): IAA high production; (++): IAA medium production; (+): IAA low production.

Focusing on the advantages of using the performed microplate assay as a routine PGPR screening method in comparison to the conventional assay, we admit that 1 ml of bacterial supernatant, and 2 ml of Salkowski reagent were used in the conventional assay. However, only 75μl of bacterial supernatant as well as 150μl of Salkowski reagent were used in the microplate assay. This outstanding reduction brings us to analyze consumption reduction in volume, cost and time of the microplate assay.

In fact, data resumed in Table 3 show a prominent reduction in bacterial supernatant volume as well as Salkowski reagent volume of about 92.5%. Furthermore, cost of chemical products was estimated (in Euro as per Sigma Aldrich ™) so that we compare the cost for each assay. It was found that the microassay is cheaper than the conventional one of around 56 %. This noticeable reduction in reagent volume is an attractive solution to reduce environmental pollution caused by liquid waste of research laboratories, that’s why; this microassay is considered as eco-friendly respecting the concept of green chemistry.

Table 3

Comparative analysis between conventional IAA assay and newly performed IAA microassay

	Conventional IAA assay		IAA microassay
	Volume/sample (ml)	Cost (€ /sample)^*	Volume/sample (ml)	Cost (€ /sample)^*
Bacterial supernatant	1	–	0.075	–
Salkowski reagent	2	0.545	0.150	0.041
Total volume (ml)	3	0.545	0.225	0.041
Incubation time (min)	30		25

^*Cost ((€ /sample): Represent the cost of the volume used per sample (for 1 test tube in the conventional assay and for 1 well in the microplate assay).

3.3 The IAA microassay meets the rules of green chemistry approach

The Environmental Protection Agency (EPA) and the American Chemical Society (ACS) have defined the concept of green chemistry as the invention, development and application of methodologies, including the possibility of applying the experiments at micro- or semi microscales, which greatly reduces the use of dangerous chemicals and sub-products, harmful to human health and environment [36, 37]. Among the 12 basic principles of green chemistry described by Anastas in 1998, the prevention and the minimization of hazardous waste production at the source was the first rule to apply by industrial companies and researchers instead of spending time and resources on managing it after the protocol is finished [37, 38]. Our performed IAA microplate assay meets exactly the green chemistry rules as it reduces, considerably the amount of Salkowski reagent waste of about 92.5%. Such reagent, mainly prepared with Sulfuric acid and Ferric chloride, is drastically toxic with skin contact and inhalation [26, 39]. Hence, the use of Salkowski reagent must be reduced in laboratory experiment to facilitate the recycling step of laboratory waste. Similarly, Abdelwahed and his collaborators performed a new colorimetric microplate assay to estimate ammonia concentration produced by PGPR basing on the minimization principle of green chemistry concept [40]. Their results show that the application of the ammonia microplate assay reduces drastically the reagent waste and toxicity hazard of K₂HgI₄ (Nessler’s reagent) in the environment of about 90%(of the total used volume in comparison to the conventional assay). Furthermore, the ammonia microassay is 10-fold cheaper and 26 times faster than the conventional method. thus, it was classified as eco-friendly respecting the Green Chemistry concept according to Environmental Protection Agency (EPA) [40].

Apart from this, the used microplate assay is 23 times faster than the traditional assay. In fact, for the conventional assay, the time needed to read the optical density of 1 strain (in triplicate) is the same time required to read the OD of 23 strains (in triplicate, with a standard range) for the microassay. Therefore, it can be seen that IAA microassay performed in 96 well microplate has several advantages as it is more economical in term of cost, time and reagent volumes when compared to the conventional assay. Figure 5 resumes a general comparison between conventional IAA assay and microplate IAA assay.

Fig. 5

General comparison between conventional IAA assay and microplate assay.

Indeed, the IAA microassay offers the ability to read the OD of all samples simultaneously since volumes of bacterial supernatants and Salkowski reagent were minimized to place the reaction mixture in 96-well flat-bottomed microplates, which reduces greatly the effort required to obtain the results. It also offers the possibility of running the experiment under the same necessary conditions such as incubation time and temperature ensured by a simple adjustment of the microplate reader parameters.

3.4 Statistical analysis

Statistical analysis was performed in order to validate the new IAA quantification assay. In fact, R free software 3.5.1 version is used to check the correlation between obtained IAA quantification results from both assays. We Used the Pearson correlation to examine the strength and direction of the linear relationship between the macro-assay (conventional assay) and the microassay data. Obtained results show very significant correlation between conventional assay and the microassay with a correlation coefficient R of 0.979 which prove that both methods give similar results. In addition to the accuracy of the microplate method, it’s far faster and easier to apply in comparison with the conventional IAA assay.

In second step, we tested Kendall correlation to measure the correlation rank of both assays. The obtained Tau value (T) is of 0.818 which prove that the agreement between the two assays is strong.

We finally checked the Spearman correlation [41] in order to validate Pearson’s correlation. The coefficient rho (ρ) is of 0.945 which ensure the strong correlation between macro-assay and micro-assay results. Figure 6 represents the correlation plot for both assays.

Fig. 6

Correlation plot of conventional IAA assay results (data$macroA) and IAA microassay results (data$microA).

When comparing conventional IAA results and microassay results, we can notice a slight augmentation in microplate results which could ensure the precision and the accuracy of the microplate assay.

4 Conclusion

The IAA microassay is considered as a reliable, rapid, cost-effective and eco-friendly method to screen plant growth promoting potential of a large number of PGP bacteria. 13 bacterial strains from different genera were used to validate the IAA microassay. Statistical analysis proves the accuracy of the microplate assay so that it could be a must-have alternative to the conventional IAA assay as a routine research tool.

Funding

This study was funded by MADFORWATER project (European Union’s Horizon 2020, WATER-5c-2015 (under grant agreement no.688320).

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Acknowledgments

The authors are grateful to ISBST, Biotechpole, Univ. Manouba, Sidi Thabet, Tunisia for providing infrastructural facilities and assistance. The authors thank for financial support the European Union in the ambit of research project MADFORWATER.

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