Isolation and characterization of novel soil- and plant-associated bacteria with multiple phytohormone-degrading activities using a targeted methodology

Ethylene (ET), salicylic acid (SA) and indole-3-acetic acid (IAA) are important phytohormones regulating plant growth and development, as well as plant-microbe interactions. Plant growth-promoting bacteria (PGPB) naturally associate with plants and facilitate plant growth through a variety of mechanisms, including the ability to modulate the concentrations of these phytohormones in planta. Importantly, the wide presence of phytohormone degradation mechanisms amongst symbiotic and other soil- and plant-associated bacteria indicates that the ability to modulate phytohormone concentrations plays an important role in bacterial colonization and plant-growth promotion abilities. Obtaining phytohormone-degrading bacteria is therefore key for the development of novel solutions aiming to increase plant growth and protection. In this paper, we report an optimized targeted methodology and the consequent isolation of novel soil- and plant-associated bacteria, including rhizospheric, endophytic and phyllospheric strains, with the ability to degrade the phytohormones, SA and IAA, as well as the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). By using an optimized targeted methodology, we rapidly isolated diverse soil- and plant-associated bacteria presenting phytohormone-degrading abilities from several plants, plant tissues and environments, without the need for prior extensive and laborious isolation and maintenance of large numbers of isolates. The developed methodology facilitates PGPB research, especially in developing countries. Here, we also report, for the first time, the isolation of bacterial strains able to concomitantly catabolize three phytohormones (SA, IAA and ACC). Ultimately, the described targeted methodology and the novel phytohormone-degrading bacteria obtained in this work may be useful tools for future plant-microbe interaction studies, and in the development of new inoculant formulations for agriculture and biotechnology.


DETAILED METHODOLOGY FOR THE ISOLATION AND CHARACTERIZATION OF NOVEL SOIL AND PLANT-ASSOCIATED BACTERIA WITH MULTIPLE PHYTOHORMONE-DEGRADING ACTIVITIES CONSIDERATIONS FOR THE SELECTION OF THE SOURCE MATERIAL
Different source materials can be used for the selection of bacteria colonizing plant tissues, structures (e.g. root nodules) and the rhizosphere ( Table 1). Most of the plant-growth promoting bacteria described in the literature are isolated from soil or the rhizosphere of selected plants. However, this can represent a limitation since their consequent application may be limited to soil, plant roots or the external surfaces of plant seeds. Alternatively, endophytic bacteria can be isolated from within plant tissues and this may present several advantages in future applications. Thus for example, plant growthpromoting endophytes can be used to inoculate plants at the flowering stage, which may lead to the bacterial colonization of the newly produced seeds (Mitter et al., 2017). This may not only lead to increased plant growth but also to an increased level of protection against some pathogens, since these endophytes may directly compete with many pathogens that are transmitted via seeds. Bacterial endophytes can also be protected from the competitive soil environment, which may impact bacterial performance (e.g. plant growth promotion abilities or degradation of xenobiotics) (Hardoim et al., 2015). Therefore, the isolation and selection of endophytes may lead to the development of more efficient inoculants. Leafassociated bacteria (epiphytes or endophytes) can also be extremely useful, especially at the level of field application. Most of these bacteria can cope with the stresses presented in the leaf environment, which most rhizobacteria cannot endure (e.g. temperature shifts, UV radiation, desiccation) (Vorholt, 2012). Leaf-associated bacteria may also directly compete with many of the plant pathogens colonizing leaf tissues (e.g. Pseudomonas syringae, Xanthomonas spp.). One of the most important aspects of these bacteria resides in the possibility of their direct application onto leaves (spraying) and their subsequent ability to colonize plant tissues. Spraying is a common agricultural practice (e.g. application of pesticides or herbicides) and may facilitate the acceptance of foliar bacterial inoculants amongst farmers.

ISOLATION OF BACTERIA: NATURAL VERSUS ARTIFICIAL SELECTION SYSTEMS
Plant and rhizosphere samples can be obtained from plants growing in wild and natural habitats (herein termed natural conditions) or by using selected soils, conditions and trap plants (herein termed artificial conditions). Each system has its own advantages and disadvantages and its use may depend on diverse factors, such as, availability of material, reagents, equipment (e.g. growth chambers and greenhouses) and time ( Table 2).

c)
Alternatively, directly dip a small section of the root (i.e. 5-10 cm) several times in a sterile 10 ml solution of 30 mM MgSO4 or PBS.

d)
Vortex the solution for 30 secs to break up any soil aggregates.

e)
Perform serial dilutions using 30 mM MgSO4 or PBS 1X. Directly use the solutions (described below) or store them at 4ºC (for up to several days) for further use.

Root and root nodule endophytes a)
Repeat steps a) of the procedure described in 1.

b)
Wash the root several times with distilled water to remove soil aggregates and rhizospheric bacteria. Repeat until the root system is free of soil particles.

c)
Surface disinfect the root tissue or the root nodule by rinsing it with 70% ethanol and then with 1% bleach. This procedure may vary depending on the plant species and age. For plants with small and thin roots (e.g. tomato) a soft surface disinfection procedure is recommended. This can be accomplished by treating the roots with 70% ethanol for 1.5 min, 1% bleach solution for 10 min and 5 consecutive washes with sterile distilled water. For harder and thicker roots (e.g. tree species) and root nodules, an increased time in the disinfection solutions is recommended (e.g. 2.5 min in 70% ethanol and 15 min in 1% bleach followed by 5 consecutive washes with sterile distilled water).

d)
Crush a small section of the root tissue (i.e. 5-10 cm long) with the help of a sterile mortar and pestle. Add 1 ml of 30 mM MgSO4 or PBS to the crushed tissue. Grind the tissue.

e)
Remove the surface disinfected root nodules (3 or 4) from roots with a sterile forceps and transfer to a sterile 2 ml tube containing 500 µl of 30 mM MgSO4 or PBS. Crush the nodules with the help of a sterile micropestle.

f)
Perform serial dilutions using 30 mM MgSO4 or PBS 1X. Directly use the solutions (described below) or store them at 4ºC (for up to several days) for further use. Falcon tube, sterile plate), using disinfected forceps. Store the tissues at 4ºC for a short period of time (up to several days).

b)
Cut small sections of shoots (i.e. 2 cm long) or leafs (i.e. 2 x 2 cm) with a sterile scalpel.

c)
Repeat step c) described in procedure 2.

d)
Alternatively, after disinfection, cut small sections (i.e. ~2 cm long) of shoot tissue and place 2 or 3 sections in sterile falcon tubes containing 5 ml of 30 mM MgSO4 or PBS.
Incubate overnight at room temperature with shaking (150 rpm). This procedure is useful for the isolation of endophytes from woody tissues that are difficult to grind. Endophytes present in tissues will be released to the liquid medium which can then be used for isolation procedures.

e)
Perform serial dilutions using 30 mM MgSO4 or PBS 1X. Directly use the solutions (described below) or store them at 4ºC (for up to several days) for further use.

FROM PLANT AND SOIL SAMPLES
This easy and targeted methodology is based on bacterial enrichment by using a minimal medium containing the selected phytohormone as the sole carbon or nitrogen source. Any of the solutions previously employed in procedures described in the previous section can be used to isolate these bacteria. Using this simple isolation technique, a wide range of phytohormone-degrading bacteria can be easily isolated.
All growth media and stock solutions used in the following section are described at the end of this document.

Isolation
Although a generic growth medium can be used, in an effort to isolate a wide range of phytohormone-degrading bacteria, it is also possible to use specific media and perform a second targeted approach to isolate particular bacterial groups. supplemented with congo red (25mg/L). Most rhizobia present whitish mucoid colonies in this medium.

Pseudomonas
Note: Additionally, plates can also be incubated at different temperatures that can promote the growth of specific bacteria (e.g. 7ºC for psychrophilic bacteria or 50ºC for thermophiles).

Determination of ACC degradation
Qualitative ACC degradation can be easily confirmed by testing the bacteria isolated (pure cultures) for its ability to grow in minimal medium containing ACC as sole nitrogen source.
The following steps should be performed in duplicate:

a)
Inoculate a colony in 5 ml DF or M9 medium containing 3 mM ACC as the sole nitrogen source (tester).

b)
Inoculate a colony in 5 ml DF or M9 medium without any nitrogen source (negative control).

c)
Incubate the inoculated media at 28ºC and 150-200 rpm for 5 days.

d)
Measure and compare the OD600 of both bacterial solutions.

e)
A positive ACC deaminase activity is found in strains that can grow on minimal medium containing ACC, but not in minimal media without nitrogen source. It is essential to test the negative control in this experiment. In some instances, some nitrogen-fixing bacteria can grow on minimal medium containing ACC as the sole nitrogen source but can be negative for ACC deaminase activity. In dubious cases a quantitative ACC deaminase activity measurement is necessary. ACC deaminase activity can be tested using a simplified version of the method described by Penrose and Glick (Penrose and Glick, 2003). This can be performed either qualitatively or quantitatively, however, qualitative determination is more accessible for the standard microbiology lab.

Induction of ACC deaminase expression a)
Grow the selected bacteria in 5 ml of a rich medium (e.g. TSB, YMB) in a 50-ml falcon tube until luxuriant growth is achieved. This depends on the bacterial strain. Usually, Pseudomonas strains grow very well in 24 h, but other strains, such as, rhizobia or some actinobacteria grow more slowly (48 to 72 h). Incubate at 28ºC, 150-200 rpm.

b)
Centrifuge the 50-ml falcon tube at 4000 rpm in a benchtop centrifuge for 10 min and discard the supernatant.

c)
Suspend and wash the cell pellet in either 5 ml DF or M9 minimal medium without a nitrogen source. Centrifuge at 4000 rpm for 10 min. Discard the supernatant.

d)
Suspend the cell pellet in either 5 ml DF or M9 minimal medium containing 3 mM ACC as the sole nitrogen source. Incubate for 24 or 48 h at 28ºC, 150-200 rpm. This step induces ACC deaminase activity. Be consistent with the incubation time.

e)
Centrifuge the tube at 4000 rpm for 10 min and discard the supernatant.

f)
Suspend the cell pellet in 1 ml 0.1 M Tris-HCl pH 8.0 and transfer it to a 1.5 ml tube.
Centrifuge the suspended cells at 10000 rpm for 1.5 min in a micro-centrifuge.

g)
Remove the supernatant and suspend the cells in 400 µl of 0.1M Tris-HCl pH 8.0.

ACC deaminase activity determination a)
Add 20 µl toluene and vortex for 30 seconds (cell permeabilization). This step is crucial for effectively measuring ACC deaminase activity. Some bacterial strains are more resistant to the procedure; in that case small glass beads can be added to the lysate (1:10 v/v) to help disrupting the bacterial cell wall and membrane. Vortex for additional 30 seconds. Note that ACC deaminase is a cytoplasmic enzyme (Jacobson et al., 1994).

b)
Dispense 50 µl of lysate into 1.5 ml centrifuge tubes: Two tubes for lysate + ACC (tester); Two tubes for lysate and no ACC (negative control). Also include an internal control: one tube containing 50 µl 0.1M Tris-HCl pH 8.0 + ACC. Save the rest of the lysate at 4ºC (for up to a few days) or -20ºC (for longer periods) for protein concentration measurements or assay repetition.

c)
Add 5 µl of 0.3M ACC to each 1.5 ml tube containing 50 µl of lysate (except for the negative controls of each sample) and vortex, approximately 5 secs.

d)
Incubate at 30°C for 30 min.

g)
Add 500 µl supernatant or standard to a glass test tube (13 x 100 mm) and then add 400 µl 0.56M HCl.

b)
Incubate at 30°C for 30 min.

c)
Add 1 ml 2 N NaOH and vortex for ~5 sec
Note: The derivatization step does not account for ACC deaminase activity. In this step, the enzyme is inactive due to the acidic pH, and the unique purpose is to derivatize phenylhydrazine to phenylhydrazone.

Protein content measurement
Measure protein content of 50 µl lysate. This can be achieved by using the Bradford reagent following the manufacturers specification and using a Bovine Serum Albumin (BSA) standard curve.

Final representation of ACC deaminase activity
The final ACC deaminase activity should be expressed in µmol a-ketobutyrate/mg protein/hour. It is calculated in the following manner: a-ketobutyrate in sample = [OD540 sample (sample + ACC)] -[OD540 negative control (sample without ACC)]. Use the a-ketobutyrate standard curve to calculate the correct aketobutyrate value.
The obtained a-ketobutyrate value is divided by the amount of protein present in the 50 µl lysate. This value is then multiplied by 2 since the assay for ACC deaminase activity was determined in only half an hour.
Alternatively, a qualitative estimation of ACC deaminase activity can be made by observing the production of a-ketobutyrate (Figure 1). This does not require either a standard curve or protein quantification.
If relative a-ketobutyrate in a sample is > 0 then the sample is positive for ACC deaminase.
Nevertheless, these values need to be interpreted carefully. Most times values close to 0 (ranging from 0 to 0.08) are deemed to represent non-specific enzymatic activities from ACC deaminase related enzymes. This occurs frequently in Enterobacteriaceae, and for example, in some Pseudomonas and Bacillus spp. which e.g. possess D-cysteine desulfhydrase.

Qualitative determination of SA and IAA degradation
Qualitative IAA or SA degradation can be easily confirmed by testing the isolated bacterial cells (pure cultures) for their ability to grow in minimal medium containing IAA or SA as a sole carbon source.
The following is typically performed in duplicate:

a)
Inoculate a small amount of a bacterial colony in 5 ml DF or M9 medium containing 1 mM IAA/SA as the sole carbon source (tester).

b)
Inoculate a small amount of a bacterial colony in 5 ml DF or M9 medium without any carbon source (negative control).

c)
Incubate the bacterial cell suspension at 28ºC, 150 rpm for 5 days (or more depending on the bacterium).

d)
Measure and compare the OD600 of both bacterial solutions, i.e. a and b above.
A positive SA/IAA degradation activity is inferred from strains that can grow on minimal medium containing SA/IAA, but not in minimal media without an added carbon source.

SA degradation test
Alternatively, the SA degradation test can be performed in 48 or 24-well plates containing minimal medium supplemented with 1 mM SA and 0.8% agar (e.g. 24-well plates = 1 ml M9 medium containing 1 mM SA as sole carbon source per well). In this case, 5 µl of an overnight grown culture (grown in general rich medium) is inoculated in the center of the plate/well. The plate is then incubated for 24-48 h at 28ºC.
SA is fluorescent under UV radiation, so, SA degradation can easily be identified by examining plates under UV radiation. A UV transilluminator, commonly used in molecular biology procedures, may be employed for this purpose. The wells inoculated with strains unable to degrade SA appear fluorescent (Figure 2A) while the wells containing strains that can degrade SA will not fluoresce ( Figure 2B).
Alternatively, the Trinder reagent (described in the supplementary information) can be added to the medium (1 ml Trinder reagent/per well of a 24-well plate) and then incubated for 20 to 30 min. The Trinder reagent is commonly used for the detection of SA (Trinder, 1954). If SA is present, the medium will change color (from yellow to purple) ( Figure 2C). Bacteria that can degrade SA remove it from the medium and hence no color development is observed ( Figure 2D).

IAA degradation test
An IAA degradation test can be performed in 48 or 24-well plates containing minimal medium supplemented with 1 mM IAA and 0.8% agar (e.g. 24-well plates = 1 ml M9 medium containing 1 mM IAA as sole carbon source per well). In duplicate, add 5 µl of an overnight culture (grown in general rich medium) in the center of the well. The plate is then incubated for 24-48 h at 28ºC.
The detection methodology is based on the use of the Salkowski reagent that is widely used in the determination of IAA production in bacterial culture medium (Glickmann and Dessaux, 1995). After incubation and growth, a solution of Salkowski reagent (described below) can be added to the solid medium (1 ml Salkowski reagent/per well of a 24-well plate) and further incubated for 1 hour at room temperature. Plates/wells containing bacteria unable to degrade IAA will change to a pink color (negative) ( Figure 3A). Bacteria able to degrade IAA will consume all of the available IAA and no color development will be observed (positive) ( Figure 3B).

Stock ACC
Stock aliquots of ACC (0.3M) can be prepared by diluting ACC in water and then filter sterilizing the diluted ACC before storing the aliquots at -20ºC (for short term use) or -80ºC (for long term storage).

Stock SA and IAA
Stock aliquots of SA (0.1M) and IAA (0.1M) can be prepared by diluting SA or IAA in a 4:1 (v:v) solution of water and 1N NaOH and then filter sterilizing the diluted phytohormones and storing them at -20ºC.

Trace elements solution:
In 100 ml sterile distilled water dissolve: H3BO3 10 mg MnSO4.H2O 11.19 mg ZnSO4.7H2O 124.6 mg CuSO4.5H2O 78.22mg MoO3 10mg Iron solution: dissolve 100 mg of FeSO4.7H2O in 10 ml sterile distilled water Final Media: Add 0.1 ml of each of the solutions of trace elements and iron to the base medium, to a final volume of 1 liter. Adjust pH to 7 with KOH.