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Eurypsychrophilic Pseudomonas spp. isolated from Venezuelan tropical glaciers as promoters of wheat growth and biocontrol agents of plant pathogens at low temperatures

  • Johnma J. Rondón
  • María M. Ball
  • Luz Thais Castro
  • Luis Andrés YarzábalEmail author
Original Article
  • 41 Downloads

Abstract

Andean tropical glaciers are disappearing rapidly and, consequently, the microbes immured in these frozen environments will be lost forever. Some of these microbes are thought to be potentially useful to develop biotechnological products or processes. Among these microbes, plant-growth promoting (PGP) bacteria have been proposed as valuable tools to develop cold-active biofertilizers and/or biopesticides. A few years ago, we hypothesized that bacteria immured within glacial ice could be effective in promoting plant growth and/or in protecting plants from pathogen infection, at low temperatures. In this study, we aimed at testing some of these traits, with a suitable plant model (Triticum aestivum). In the present study, from a collection of bacteria isolated from Venezuelan tropical glaciers, we selected four Pseudomonas isolates and tested their PGP effects at low temperatures, both in vitro and on wheat plantlets. The isolates grew well over a wide range of low temperatures and were thus classified as eurypsychrophilic. They also displayed well-known PGP traits: solubilization of inorganic phosphates, production of phytohormones and antagonism against a phytopathogenic oomycete (Pythium ultimum). Inoculation of T. aestivum seeds with some of these Pseudomonas spp. isolates promoted a significant elongation of their roots and shoots. This was also the case when wheat plantlets were grown in sterile sand or soil, at 15 °C. Inoculation of wheat seeds also protected plantlets against the damage caused by P. ultimum. Together, our results suggest that some of these Pseudomonas spp. isolates could act as cold-active biofertilizers and/or biocontrol agents.

Keywords

Andes Mountains Tropical glaciers Plant-growth promoting bacteria Pseudomonas Eurypsychrophiles Biocontrol agents 

Introduction

Tropical glaciers are massive repositories of a virtually unexplored biologic-, physiologic-, and genomic diversity (Edwards 2015). Unfortunately, most of them are retreating rapidly and will disappear in the next few years (Rabatel et al. 2013; Vuille et al. 2018). This is the case of Venezuelan tropical glaciers, which have lost more than 90% of its surface area during the last 50 years (Braun and Bezada 2013). These glaciers are amongst the fastest melting glaciers in the entire Andean region and will probably disappear in less than 5 years.

For these reasons, in 2010 we began studying the microbial diversity of the last two Venezuelan glaciers with the aims of characterizing the microbial communities immured within them, isolating and preserving as many microbial strains as possible and, most importantly, identifying potentially useful microbes to develop biotechnological processes and products.

Among the microbes found in glacier environments, bacteria belonging to the genus Pseudomonas stand out as they have been reported to be metabolically diverse, tolerant to different kinds of stress, adaptable to a wide range of environmental conditions, and efficient colonizers of the rhizosphere of many plants, where they could play important roles in plant nutrition and protection (Lugtenberg et al. 2001; Raaijmakers et al. 2002; Spiers et al. 2000; Silby et al. 2011; Wu et al. 2011).

To date, only a few psychrotolerant and psychrophilic Pseudomonas strains have been shown to promote plant growth and development at low temperatures (Daayf et al. 2003; De Curtis et al. 2010; Haas and Défago 2005; Kim and Jeun 2006; Scherwinski et al. 2008; Yan et al. 2002). Recently, one of us showed that eurypsychrophilic Pseudomonas strains, isolated from Antarctic soils, promote wheat germination and growth at cold temperatures (Yarzábal et al. 2018), confirming their biotechnological potential to develop cold active biofertilizers.

However, to the best of our knowledge, it has not been tested to date if bacteria immured in tropical glaciers, for long periods, can be effective in promoting plant growth and/or in protecting them from pathogen infection at low temperatures. A few years ago, we hypothesized that this could be the case (Balcázar et al. 2015). Therefore, the main objective of this work was to test several Pseudomonas spp. strains, isolated from tropical Venezuelan glaciers located at high altitudes, for their abilities to promote wheat growth and development at low temperatures. We also aimed at testing the protective effect of these bacterial isolates on wheat plantlets infected with a phytopathogenic oomycete.

Materials and methods

Sampling sites and isolation of bacteria

Bacteria were isolated from two Venezuelan Andean glaciers as previously described in Ball et al. (2014) and Rondón et al. (2016). Bacterial colonies were selected based on their morphological differences, further purified until obtaining clonal populations of each isolate, and finally stored at − 80 °C in 20% glycerol.

Identification of isolates

PCR amplification of an internal fragment of the 16S rDNA gene of each bacterial isolate was performed as previously described (Rondón et al. 2016). The PCR products were purified with the Wizard SV PCR clean up system kit (Promega, Wisconsin USA) and sequenced by automated Sanger sequencing at Macrogen (Seoul, Korea). Once edited and assembled, the nucleotide sequences were compared for similarity against sequences deposited in the GenBank, using NCBI-BLAST (Altschul et al. 1997). To assign each sequence to a specific taxon, the closest match of known phylogenetic affiliation was used. All the partial 16S rDNA sequences generated in the study were deposited in GenBank with the accession numbers presented in Table 1.
Table 1

Characteristics of Pseudomonas isolates

Isolate

Genbank accession number

Closest related species and strain (blast search)

16S rDNA similarity (%)

Generation time (h)

Maximum specific growth rate (h−1) in R2A

Maximum cell density (UFC/ml at stationary phase in R2A)

PGV045

KM114891

P. orientalis

99

33.81

0.0205

1.19 × 109

PGV094

KF020685

P. brenneri

99

25.39

0.0273

1.2 × 109

PGV233

KJ417592

P. antarctica

99

29.62

0.0234

1.18 × 109

PGV274

MH824582

P. brenneri

86

30.67

0.0226

1.2 × 109

Characterization of Pseudomonas spp. isolates

The kinetics of growth of a few selected Pseudomonas spp. isolates was determined by measuring the absorbance at 600 nm of cultures grown at 15 °C, with occasional agitation, in R2A medium (Reasoner and Geldreich 1985).

Monitoring bacterial PGP traits

In order to determine their ability to solubilize inorganic-P containing minerals, Pseudomonas spp. isolates were grown in a double layer agarized medium (NBRIP) (Nautiyal 1999) at 14 °C. This medium contained Ca3(PO4)2 (tricalcium phosphate or TCP) as sole source of P, only on the top layer. Plates were visually inspected daily until observing the formation of a clear halo surrounding the colonies, indicative of TCP solubilization.

In addition, the concentration of soluble P in culture supernatants was estimated as described by Balcázar et al. (2015). Briefly, 20 ml liquid NBRIP were inoculated with approximately 108 cells/ml of each bacterial isolate, and incubated at 15 °C for 7 days with occasional agitation in presence of glucose (1%) as sole carbon source. To perform this test, different sources of insoluble P were tested: TCP, iron phosphate (FePO4 or FeP) and aluminum phosphate (AlPO4 or AlP) at a final concentration of 1 g/l. The concentration of soluble P was estimated in the culture supernatants by means of the vanado-molybdate blue method (Murphy and Riley 1962).

The concentration of d-gluconic acid in the culture supernatants was estimated using an enzymatic detection kit (Megazyme, product K-GATE), following the manufacturer’s instructions. On the other hand, the production and secretion of indoleacetic acid (IAA) by Pseudomonas spp. isolates was tested on tryptophan-supplemented (5 mM) R2A medium, using the rapid in situ bioassay developed by Bric et al. (1991). Finally, the production of siderophores was monitored following the Chrome Azurol-S (CAS) method (Schwyn and Neilands 1987), whereas HCN was detected by the method of Bakker and Schippers (1987).

In vitro assessment of the antagonistic activity of isolates

The antagonistic activity of Pseudomonas spp. isolates was evaluated against Pythium ultimum strain 67-1, using two different assays:

Dual culture assay

From a 7-day old culture of P. ultimum in potato dextrose agar (PDA), discs were excised using a 5 mm biopsy punch, and placed in the center of fresh PDA plates. Then, actively grown cells of each bacterial isolate were spotted in the periphery of the same plates, 3 cm away from the agar disc. Plates were incubated at 25 °C (to allow P. ultimum growth) and the inhibition of oomycete growth in the proximity of bacterial colonies was recorded by visual inspection every 24 h after inoculation, up to 12 days. As a positive control of P. ultimum growth inhibition, CHA0 strain of Pseudomonas protegens, a very efficient biocontroller, was taken (Haas et al. 2002; Ramette et al. 2011).

Volatile compound assay

The inhibitory effect of volatile metabolites produced by Pseudomonas spp. isolates on oomycete growth was assessed by means of the dual plate bioassay of Alström and Burns (1989). As described above, a 7-day old 5 mm diameter agar disc—containing an active mycelium of P. ultimum—was placed in the center of PDA. The mycelium was allowed to grow at 25 °C for 3 days. Simultaneously, each Pseudomonas spp. isolate was streaked on the surface of Nutrient Agar, and incubated for 48 h at 15 °C. The lower part of the plates containing the oomycete mycelium and the bacterial lawn were paired together (mycelium on top), sealed with parafilm and incubated at 25 °C for 7 days, to monitor for inhibition of mycelial growth. Again, the positive control of inhibition was performed using P. protegens CHA0, a producer of a toxic mixture of volatile metabolites. The experiment was conducted in triplicate.

Compatibility test

In order to test the compatibility between the Pseudomonas isolates, 5 µl samples collected from liquid cultures of each strain (producers) were spotted on the surface of R2A agar medium and incubated at 15 °C for 48 h. Then, 0.2 ml samples of each strain (reporters) were mixed with 3 ml of molten R2A agar medium (containing 7.5 g/l agar) and spread over the surface of the medium, where producers had already grown. The plates were incubated for another 48 h at 15 °C, and photographed to record any inhibition halo of the reporter strain that formed around producer colonies. The assay was performed in triplicate.

Promotion of wheat growth by Pseudomonas spp. isolates

Primary root and shoot growth elongation test

The rolled paper towel bioassay developed by Mishra et al. (2008) was used to determine the effect of Pseudomonas spp. isolates on primary root- and shoot elongation of wheat. First, the seeds were inoculated following the protocol developed by Lifshitz et al. (1987), with a few modifications. In brief, approximately 200 seeds were soaked twice in 50 ml 3% sodium hypochlorite, for 10 min at room temperature (~ 20 °C), to sterilize their surface. Then, the seeds were washed five times with sterile distilled water, to remove any traces of bleach. Once sterilized, the seeds were deposited on sterile Petri dishes (approximately 100 seeds per plate), covered with 3 ml bacterial suspensions (~ 108 CFU/ml) and incubated inside a laminar flow hood, until the liquid medium dried completely. The bacteria-coated seeds were aseptically distributed on the surface of a sterile paper towel sheet, which was then rolled and placed inside a 1 l beaker. Seeds treated exactly the same way with sterile liquid medium were included as negative controls. Hoagland’s nutrient solution (Hoagland and Arnon 1938) was used to moisten the rolled paper. At the end of an 8-day period of incubation at 15 °C ± 1 °C, the length of the primary roots and shoots was measured.

The vigor index (VI) was calculated according to the formula of Abdul-Baki and Anderson (1973):
$${\text{VI}} = {\text{SG}} \times \left( {{\text{SL}} + {\text{RL}}} \right),$$
where, SG is the seed germination (%), SL is the mean shoot length (cm), RL is the mean radicle length (cm).

Plant growth promotion test

Substrate—Sterile sand: A pot assay-based determination of the PGP abilities of the Pseudomonas spp. isolates was conducted, as described in Yarzábal et al. (2018). In brief, wheat seeds were sown in sterile sand under growth chamber conditions, and in the presence of a barely soluble form of P, namely TCP. The seeds were first bacterized as specified above and then sown in polystyrene cups (8.0-cm diameter and 10-cm depth) containing 100 g of sterile sand. For the negative control experiment, seeds were coated with sterile medium. The cups were placed in a temperature controlled growth chamber, in a completely randomized design with five replications, and incubated at 15 ± 2 °C for 16 days with a day length of 12 h, using fluorescent lamps. The sand was moistened to 60% of its water-holding capacity with Hoagland’ solution, containing TCP as sole source of P. Each cup was re-inoculated after 8 days with 4 ml of a pure culture of each bacterial strain (109 CFU/ml). At the end of the experimental period, the plantlets were uprooted, and washed under running water, before measuring their roots and shoots.

Substrate—Sterile soil: The assay was conducted essentially as described above, but this time using sterile soil (Supplementary Table S1). Again, a completely randomized design, with five replications, was followed to arrange the treatments. The control experiment consisted of non-inoculated seeds, treated exactly the same way. After 16-days’ growth, the plantlets were uprooted and washed under running water, in order to measure the lengths of their roots and shoots. The same assay was conducted in the presence of different combinations of bacterial strains, combined pairwise based on the results obtained in the compatibility assay (see Sect. “Compatibility test”).

Bioprotection of wheat seedlings against P. ultimum infection

In order to test the protective effect of Pseudomonas spp. against the damage caused to wheat plantlets by P. ultimum infection, we conducted another pot assay essentially as described above (see Sect. “Plant‑growth promotion tests”). However, in this case, the plantlets were re-inoculated with 3 ml of each bacterial suspension (108 CFU/ml) after 7 days of growth, and were challenged the next day with a 4 ml inoculum of P. ultimum (approximately 104 propagules/ml). Again, at the end of the experimental period, the root- and shoot lengths of the plantlets were measured. The control experiment consisted of plantlets that were not protected against the pathogen, by inoculation with the Pseudomonas spp. isolates.

Statistical analyses

One-way ANOVA with post hoc Dunn or Dunnett’s tests were used for statistical comparison of the data recorded (i.e. primary root- and shoot- length of seedlings; root- and shoot lengths of wheat plantlets subjected to different treatments).

Results

Characterization of isolates

The bacterial isolates included in this study belong to the Pseudomonas genus, as determined by 16S rDNA sequence analyses (Table 1). Incidentally, the closest phylogenetic neighbors were, in all four cases, bacterial strains isolated from glacial- or permanently cold environments. The colony morphology of the isolates was similar (i.e. white, smooth and brilliant); however, two of them (PGV094 and PGV274) exhibited a highly mucoid aspect, mainly when growing at lower temperatures (i.e. at or below 15 °C).

The bacterial isolates were classified as eurypsychrophilic since they were able to grow over a wide range of low temperatures (i.e. from 4 °C to 30 °C). The kinetics of growth at 15 °C (of the isolates) are shown in Fig. 1 and the growth parameters are presented in Table 1. As can be seen, the isolates showed similar growth kinetics, attaining the stationary phase at approximately 80 h, and exhibiting high cell densities (> 109 CFU/ml).
Fig. 1

Growth kinetics of Pseudomonas spp. isolates in R2A medium. Incubation was done at 15 °C for 5 days

Plant-growth promotion traits of isolates

Mineral phosphate solubilization assays

Quantitative estimation of the P-solubilization activity of the Pseudomonas spp. isolates, conducted in liquid NBRIP medium, revealed that all of them were able to solubilize TCP at 15 °C in the presence of glucose (Fig. 2a). The P-solubilizing ability exhibited by the isolates depended on the chemical form of the available mineral-P. Indeed, the order of solubilization in the presence of soluble bacterial metabolites was TCP ≫ AlP > FeP (Fig. 2a, b).
Fig. 2

Bacterially-mediated mineral phosphate solubilization. aPseudomonas spp. isolates were incubated for 7 days in NBRIP medium at 15 °C, containing tri-calcium phosphate as sole source of P, in the presence of different sugars as sole sources of C and energy (see “Materials and methods”). b Solubilization of FeP and AlP mediated by Pseudomonas spp. isolates in NBRIP medium containing glucose (as sole source of energy and C). Incubation at 15 °C for 7 days

Gluconic acid production

All the Pseudomonas spp. isolates produced gluconic acid from glucose in liquid NBRIP medium, at 15 °C. As can be seen in Fig. 3, gluconic acid accumulated in the supernatant of cultures, attaining in some cases a concentration higher than 2.2 g/l.
Fig. 3

Gluconic acid production by Pseudomonas spp. isolates. Bacterial isolates were grown in NBRIP medium for several days at 15 °C and gluconic acid concentration quantified as specified in “Materials and methods”

Production of phytohormones and antagonistic metabolites

Aside from solubilizing inorganic forms of P, in the presence of different sugars and at 15 °C, the Pseudomonas spp. isolates synthesized IAA in tryptophane-supplemented R2A medium, as revealed by the production of a pink-colored product (Fig S1A supplementary).

The isolates also synthesized siderophores (Fig S1B supplementary). In line with this result, the isolates inhibited growth of P. ultimum likely because they produced soluble inhibitory compounds, as revealed by dual culture assays (Table 2 and Fig. S2 Supplementary). The isolates also produced a mixture of volatile compounds able to inhibit, totally or partially, radial growth of the oomycete (Table 2 and Fig. S3 supplementary). In some cases, the inhibition was greater than the one manifested by P. protegens CHA0. HCN was detected among these volatile metabolites, but only in the case of isolate PGV094.
Table 2

Antagonistic behavior of psychrophilic Pseudomonas spp. isolates against P. ultimum

Isolate

P. ultimum inhibition assay (volatiles)

P. ultimum inhibition assay (soluble)

PGV045

**

++

PGV094

*

++

PGV233

*

++

PGV274

***

++

P. protegens CHA0

*

+++

++: inhibition halo diameter < 4 mm; +++: inhibition halo diameter > 4 mm

*Medium growth; **low growth; ***no growth

Plant-growth promotion tests

Germination tests

No significant differences were found when comparing the percentage germination of wheat seeds inoculated or not with Pseudomonas spp. isolates (p = 0.153; Table 3). The same was true when comparing the radicle length or the shoot length of the seedlings. Surprisingly, isolate PGV274 negatively affected root growth of wheat seedlings. Even though some differences were apparent when considering the germination vigor, they were not statistically significant (Table 3).
Table 3

Effect of psychrophilic Pseudomonas spp. inoculation on germination vigour of wheat seeds

Treatment

% germination of seeds

Radicle length (cm)

Shoot length (cm)

Germination vigour

Control

98.3 ± 1.4

5.6 ± 1.4

2.2 ± 0.6

774.6

PGV045

99.2 ± 1.3

5.9 ± 1.7

2.1 ± 0.8

798.5

PGV094

94.7 ± 1.2

7.1 ± 1.3

2.6 ± 1.1

920.5

PGV233

98.1 ± 3.2

6.1 ± 1.1

2.2 ± 0.7

824

PGV274

99.1 ± 1.3

3.8 ± 1.6*

2.2 ± 0.6

608.4

ANOVA post hoc Dunnet’s test *p < 0.05. Results are mean of 05 replicates ± standard deviation (SD)

Wheat-growth promotion assay

Based on the combined results obtained in the previous assays, we tested the PGP ability of Pseudomonas spp. isolates—alone or in combination with other isolates—on wheat, in pot assays under sterile substrate conditions.

After 16 days’ growth at 15 °C in sterile sand in the presence of a barely soluble form of P (TCP), bacterized wheat seedlings attained greater root- and shoot lengths than non-inoculated ones (Table 4). In the case of isolate PGV045, the shoot length of the plantlets was on average 51.8% greater than the non-inoculated controls (p < 0.005); a similar observation was made when focusing at the roots of bacterized plantlets, which were on average 233.3% greater than the ones belonging to the control plantlets (p < 0.05). Similar results were obtained with strains PGV094 and PGV233. Surprisingly, isolate PGV274 seemed to negatively affect plantlet growth, since the shoot lengths of plantlets inoculated with this strain were 48.1% shorter than those belonging to the control plants (p < 0.05).
Table 4

Effect of psychrophilic Pseudomonas spp. inoculation on wheat growth (shoot and root length) in sterile sand- and sterile soil microcosms at 15 °C

Treatment

Sand microcosms

Soil microcosms

Shoot length (cm)

Root length (cm)

Shoot length (cm)

Root length (cm)

Control

11.0 ± 6.4

1.8 ± 0.5

22.0 ± 6.1

10.2 ± 2.5

PGV045

16.7 ± 3.6**

6.0 ± 1.9*

24.3 ± 3.4

13.7 ± 2.9*

PGV094

16.6 ± 4.1**

5.2 ± 1.9*

19.9 ± 8.8

9.4 ± 4.3

PGV233

17.5 ± 3.7***

3.2 ± 1.6

21.1 ± 5.5

10.8 ± 1.9

PGV274

5.7 ± 2.9*

1.9 ± 0.6

21.5 ± 4.2

13.6 ± 3.6*

ANOVA post hoc Dunn’s test *p < 0.05; **p < 0.005; ***p < 0.001. Results are mean of 05 replicates ± SD

When the growth promotion assays were conducted in sterile soil microcosms, no significant differences were observed when comparing the shoot length of inoculated and non-inoculated plantlets (Table 4). However, roots of plantlets inoculated with isolates PGV045 and PGV274 were significantly longer (p < 0.05) than the roots of uninoculated control plants. Contrary to what we observed in sand microcosms, strain PGV274 did not exert a negative effect on plantlet growth.

In order to test a potential synergistic effect between the Pseudomonas spp. isolates, and considering that no inhibition was observed when co-culturing them in the compatibility tests (data not shown), the strains were inoculated pairwise into the soil-microcosms. This time, the results were markedly different to those commented above. For instance, the combinations PGV045/PGV233 and PGV045/PGV274 promoted a significant increase in the development of the plantlet shoots, as compared to the uninoculated controls (p < 0.05) (Table 5). However, no differences were found concerning the root lengths, in this assay.
Table 5

Effect of different combinations of psychrophilic Pseudomonas spp. on wheat growth (shoot and root length) in sterile soil microcosms at 15 °C

Treatment

Shoot length (cm)

Root length (cm)

Control

15.8 ± 3.6

10.9 ± 2.9

PGV045 + PGV094

19.1 ± 5.2

11.7 ± 1.6

PGV045 + PGV233

19.0 ± 4.5

10.8 ± 3.2

PGV045 + PGV274

19.8 ± 1.9*

11.1 ± 2.2

PGV094 + PGV233

19.2 ± 2.2*

11.1 ± 2.1

PGV094 + PGV274

18.0 ± 4.4

6.5 ± 3.5*

PGV233 + PGV274

17.7 ± 7.4

7.3 ± 3.6

ANOVA post hoc Dunn’s test *p < 0.05. Results are mean of 05 replicates ± SD

Wheat plantlet protection by Pseudomonas spp. against phytopathogen

In order to test whether the antagonistic behavior of Pseudomonas spp. isolates against P. ultimum detected in vitro (see above, Sect. “Production of phytohormones and antagonistic metabolites”) would protect wheat plantlets against the damage caused by P. ultimum, bioprotection assays were conducted in pots containing sterile soil. The results presented in Table 6 clearly show that bacterization of seeds protected the plantlets against P. ultimum infection. In general, inoculation with each one of the strains allowed the plantlets to reach greater shoot- and root-lengths (and a better general aspect) than the ones recorded in the control experiments. The only exception to this trend were isolates PGV045 and PGV233, which did not seem to protect the roots of wheat plantlets, even though the shoot of the same plants seemed to be unaffected by P. ultimum.
Table 6

Protective effect of psychrophilic Pseudomonas spp. against P. ultimum infection and damage of wheat seedlings grown at 15 °C

Treatment

Shoot length (cm)

Root length (cm)

Control

16.38 ± 6.19

6.56 ± 1.61

PGV045

24.99 ± 3.12**

8.16 ± 2.12

PGV094

21.12 ± 5.86*

9.99 ± 2.77+

PGV233

25.22 ± 5.54**

10.72 ± 4.24

PGV274

24.19 ± 6.87**

10.3 ± 4.45+

P. protegens CHA0

23.81 ± 4.06**

9.21 ± 2.22+

ANOVA post hoc Dunnett’s test *p < 0.05; **p < 0.001

ANOVA post hoc Dunn’s test +p<0.05. Results are mean of 05 replicates ± standard deviation SD

Discussion

It is widely acknowledged that members of the genus Pseudomonas act as plant growth promoting bacteria (Antoun and Prévost 2006; Vacheron et al. 2013). Recently, we showed that eurypsychrophilic Pseudomonas strains, isolated from Antarctic soils, not only exhibited PGP-abilities in vitro, but promoted wheat germination and growth at low temperatures (Yarzábal et al. 2018). Here we show, for the first time, that Pseudomonas isolates—immured for many years in glacial ice—can not only enhance growth of wheat plantlets at 15 °C, but also protect them against the damage caused by a phytopathogen like P. ultimum.

The Pseudomonas spp. isolates studied were able to grow at a wide range of temperatures, from 4 to 30 °C. At 15 °C, the cultures reached cell densities comparable to the ones exhibited by their mesophilic counterparts. Even though for a long time they were considered as mesophiles (i.e. organisms with an optimal growth temperature in the range 20–40 °C), nowadays there is general consensus that some Pseudomonas spp. strains are psychrotolerant and even bona fide psychrophiles. There is also general agreement that the ecological success of Pseudomonas spp. under harsh natural conditions is a consequence of their metabolic diversity and their ability to cope with low temperatures and other types of stress (Moreno and Rojo 2014).

In the past, many psychrophilic or psychrotolerant strains of Pseudomonas spp. have been shown to promote growth and development of several crops (Katiyar and Goel 2003; Egamberdiyeva and Höflich 2003; Pandey et al. 2006; Trivedi and Pandey 2007; Kumar et al. 2007; Trivedi et al. 2007; Trivedi and Sa 2008; Mishra et al. 2008, 2009, 2011; Selvakumar et al. 2009, 2011; Bisht et al. 2013). The majority of these assays were conducted by Indian researchers, working with Pseudomonas strains isolated from the Himalayan Mountains. Some of these strains were even used to develop low cost- biofertilizers, particularly useful to substitute toxic agrochemicals (Paulin and Filion 2013).

Antarctic Pseudomonas strains are also able to enhance plant growth; this is the case, for instance, of strain Da-Bac, which promoted growth of Deschampsia antarctica under controlled conditions (Berríos et al. 2013). Eight Antarctic Pseudomonas strains, isolated and characterized by Yarzábal et al. (2018), were also shown to promote wheat germination and growth at 14 °C, in addition to antagonize fungal plant pathogens in vitro. In the present work, we extend further the knowledge concerning the PGP-abilities of eurypsychrophilic Pseudomonas strains showing that, aside from promoting wheat growth at low temperatures under controlled conditions, they can also protect plantlets against the damage caused by a pathogenic oomycete.

Stimulation of plant growth by bacteria is the consequence of both direct- and indirect mechanisms (Persello-Cartieaux et al. 2003; Vessey 2003; Glick 2012). The PGP-effect manifested by the Pseudomonas spp. isolates characterized here is likely due to several mechanisms acting simultaneously: production of phytohormones (like IAA), solubilization of mineral phosphates, and production of a mixture of inhibitory substances (like siderophores), which prevents growth of pathogens.

Among the above mentioned mechanisms, one of them is of particular importance: the solubilization of inorganic phosphates. Indeed, phosphorus—the most important macronutrient for plant growth after nitrogen—is not readily available in the soil environment because it becomes rapidly fixed under the form of barely soluble Fe- and Al-phosphates. For that reason, even though it can be present in high amounts in the soil, P is frequently unavailable to plants (Schachtman et al. 1998). That is why any chemical or biological mobilization of these sparingly soluble forms of P can significantly promote plant growth.

The solubilization of mineral phosphates (TCA, Fe–P and Al–P) mediated by all four Pseudomonas isolates is almost certainly related to the production and release of high amounts of gluconic acid. This is one of the most important final products of the direct oxidation of glucose, a metabolic pathway characteristic of Pseudomonads (Lessie and Phibbs 1984). The fact that this solubilization occurred at low temperatures (i.e. 15 °C) suggests the presence of an enzymatic complex that can perform well under harsh environmental conditions, close to the extreme. As highlighted by Yarzábal (2014), this is one of the traits that should be prospected when searching for microorganisms to develop biotechnological products, like bioinoculants, effective at low temperatures.

In addition to the availability of mineral nutrients, growth and development of plants also rely on the action of phytohormones. For instance, auxins like IAA stimulate both seed- and tuber germination, aside from promoting plant-cell division, and controlling vegetative growth, among others (Glick 1995; Tsavkelova et al. 2006; Spaepen and Vanderleyden 2011). IAA is also able to enhance growth of primary roots and is a key regulator of lateral root formation, acting in a concentration-dependent manner (Patten and Glick 2002). Many bacteria colonizing plant roots, among which Pseudomonads, produce this kind of molecules (Vessey 2003; Preston 2004; Trivedi and Pandey 2007; Mishra et al. 2008, 2009; Selvakumar et al. 2009, 2013; Bisht et al. 2013; Berríos et al. 2013; Yadav et al. 2015a, b; Balcázar et al. 2015; Yarzábal et al. 2018). Our results are in line with such observations, and show that bacterization of wheat seeds with the Pseudomonas isolates characterized here resulted in an increased elongation of primary roots at low temperatures.

Surprisingly, even though the Pseudomonas spp. isolates clearly promoted wheat growth at 15 °C in pot assays containing sterile sand supplemented with TCP, they did not perform at their best when the assays were conducted in sterile soil. Among the reasons that can explain this contradictory observation, we believe that the amount of P already present in soil was so low that it could have limited plantlet growth; it is also possible that P would have been present under more insoluble forms than TCP.

On the other hand, when the isolates were inoculated pairwise in different combinations, a completely different figure emerged. Indeed, all combinations tested, significantly enhanced shoot growth of the plantlets, although they exerted almost no effect on root elongation. The former can be the result of a synergistic effect between the bacterial isolates, although from an unknown nature. One of such kind of positive interactions has been shown to occur between different strains of Pseudomonas fluorescens in soil microcosms (Luján et al. 2015). Even though the cooperation in this case was related to the production of siderophores, it was clear that the presence of one P. fluorescens strain stimulated growth of the other strain. Furthermore, it has been recently shown that interactions between P. fluorescens and other bacteria increase colonization of the wheat rhizosphere and rhizoplane, significantly enhancing its vegetative growth and improving some photosynthetic parameters (Ansari and Ahmad 2019). Therefore, social interactions between strains of Pseudomonads effectively improve their PGP-abilities. Whatever the case may be, our results are in line with previous reports (Trivedi and Pandey 2007; Trivedi and Sa 2008; Mishra et al. 2008, 2009; Selvakumar et al. 2009, 2011; Yarzábal et al. 2018) and confirm that, under certain conditions, inoculation of wheat with Pseudomonas strains can promote plant growth.

Among the most important findings of the present work, we should highlight the protection of wheat plantlets by Pseudomonas spp. isolates, against the damage caused by P. ultimum infection. This oomycete is the etiological agent of the root rot of wheat, producing brown necrotic lesions, but also causing aboveground symptoms like stunting, reduced tillering, chlorosis, and delayed maturity (Weller and Cook 1986). As shown here, when plantlets grew in the presence of Pseudomonas spp., their shoots and roots developed better and attained greater lengths than the uninoculated controls, even when challenged with an active mycelium of the pathogen. According to some authors, the antagonism exhibited by Pseudomonads against phytopathogens illustrate their potential to develop biocontrol products that may reduce the use of agrochemicals (Compant et al. 2005). In fact, Pseudomonas spp. strains are acknowledged as efficient biocontrol agents of plant pathogens (Haas and Défago 2005). This protective effect has been confirmed in assays conducted in the field (Weller and Cook 1986; Milus and Rothrock 1997; Meyer et al. 2010; Mavrodi et al. 2012).

Most likely, the protection manifested here by the bacterial isolates is related to the production of some inhibitory compounds, both soluble and volatile, as shown in the co-cultivation assays. It is well known that Pseudomonads antagonize phytopathogens and other microorganisms by producing a wide array of toxic compounds, such as antibiotics, hydrolytic enzymes and volatiles like hydrogen cyanide (Raaijmakers et al. 2002; Haas and Défago 2005). Among such inhibitory substances siderophores stand out. These low molecular-weight compounds produced by many bacteria are very efficient at scavenging iron from the environment and keeping it away from any competitor (Hider and Kong 2010). That is why siderophores are considered as biological weapons involved in inter-species competition for nutrients (Niehus et al. 2017).

Among some limitations of our work, we are aware that the PGP- and biocontrol abilities of the Pseudomonas isolates characterized here must be evaluated in plants at later stages of development, in order to clearly establish their ecological roles. It can also be argued that the stimulating effect of Pseudomonas isolates should have been studied under less controlled conditions (i.e. in the greenhouse or in the field) and with plants growing in non-sterile soils, i.e. in the presence of native communities of microorganisms. Clearly, aside from measuring the length and weight of roots and shoots, other plant parameters (like protein and chlorophyll contents, among others) must be monitored, in order to thoroughly characterize the PGP-abilities of these bacterial isolates. These, however, are the next logical steps we plan to follow, after having confirmed that the Pseudomonas isolates behave as we hypothesized.

As a final point, it is tempting to discuss how microbes, not supposed to be there, did reach the top of Andean mountains and became immured inside glacial ice? According to some authors, glaciers are repositories of microbial forms of life that were transported there, from distant areas, by winds (for instance, from the Amazon forests or the exposed soils from the Pacific coast) (Priscu and Christner 2004; Priscu et al. 2007). On the other hand, many of the culturable bacteria isolated from glacial ice cores grow at a wide range of temperatures (i.e. they are eurypsychrophiles); therefore, this trait is considered an important strategy for surviving the transition from source habitat to glacier ice (Liu et al. 2019). Nevertheless, it is also plausible that, once entrapped inside ice crystals, these microbes were not only able to survive, but also to adapt, to proliferate and even to evolve inside glacial ice, remaining viable for thousands or even millions of years (Bidle et al. 2007; D’Elia et al. 2008). Therefore, strange as it may seem at first sight, it should come as no surprise to find eurypsychrophilic PGP bacteria in Andean glacial ice.

In conclusion, as shown here for the first time, eurypsychrophilic Pseudomonas spp. isolates—immured for many years in tropical glaciers’ ice—promoted germination and growth of wheat plantlets, at low temperatures. They also protected young plantlets from the attack of a phytopathogenic oomycete, P. ultimum. These abilities were related to the production of phytohormones (like IAA) and toxic molecules (both soluble and volatile). Consequently, this unexpected microbiological resource can be exploited to develop efficient bioinoculants, able not only to promote plant growth and to antagonize phytopathogens, but also to remain active at low temperatures (Pandey et al. 2004; Trivedi et al. 2005; Yarzábal 2014). Such kind of bioinoculants are of paramount importance to develop new-, environmentally friendly solutions for mountain agriculture.

Notes

Acknowledgements

Dr. Silvia Restrepo (Universidad de Los Andes, Colombia) is grateful acknowledged for its help in sequencing some of the 16S rRNA genes presented in this manuscript. We also thank Dr. Stéfano Torracchi for its assistance in producing some of the graphics included in this work. We are grateful to Dr. Eduardo Chica for its comments and suggestions. We finally thank our colleagues from the following laboratories: Laboratorios de Enzimología de Parásitos, Laboratorio de Fitobiotecnología y Laboratorio de Biología de Parásitos de la (Facultad de Ciencias, Universidad de Los Andes, Venezuela). This work was partially financed by Fondo Nacional de Ciencias, Tecnología e Innovación (FONACIT) Project No. 2011001187. JR was the beneficiary of a ULA Plan II scholarship (Universidad de Los Andes, Venezuela).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

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Supplementary material 1 (DOCX 12 kb)
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© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Laboratorio de Microbiología Molecular y Biotecnología, Facultad de CienciasUniversidad de Los AndesMéridaVenezuela
  2. 2.Laboratorio de Fitobiotecnología, Facultad de CienciasUniversidad de Los AndesMéridaVenezuela

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