Introduction

Plant growth-promoting rhizobacteria (PGPR) in the rhizosphere can have beneficial effects on plants (Vessey 2003) by directly affecting growth through the production of plant hormones and increasing nutrient availability or indirectly by inhibiting pathogens (Richardson et al. 2009; Ahangarar et al. 2012; Zhang et al. 2014; Pii et al. 2015). Microorganisms that can promote growth of plants of agricultural interest have been widely tested. Species such as maize, tomato and cereals (Matiru and Dakora 2004; Pii et al. 2016) have shown positive responses to inoculation with PGPR, such as increased biomass, improvement of fruit quality and resistance to pathogens such as Fusarium, Phythium and Phytophthora (Whipps 2001; Zehnder et al. 2001). However, in tree species, particularly in coniferous species, studies related to inoculation with PGPR are limited (Enebak et al. 1998; Bent et al. 2001; Barriuso et al. 2005; Brunetta et al. 2007; de Vasconcellos and Cardoso 2009; Singh et al. 2010). The benefits provided by the PGPR to the host include greater nitrogen and phosphorous contents (Rojas et al. 2001; Bashan and Holguin 2002; Holguin et al. 2003; Anand et al. 2013) and an increase in biomass (Chanway and Holl 1992) and the production of organic acids, phenolic compounds and siderophores (Calvaruso et al. 2006) that help in the release of nutrients. In forest ecosystems, the PGPR affect processes leading to the release of low-availability nutrients, such as phosphorus (Calvaruso et al. 2006; Ouahmane et al. 2009). They also induce growth of plants by releasing growth factors (Probanza et al. 2002) and promoting resistance to environmental stress (Rincón et al. 2008; Sharma and Rai 2015).

In Mexico, deforestation and fires have generated losses in forest areas (Cuevas-Guzmán et al. 2011). The regeneration of forest resources is gradual, mainly because of the slow growth rate of forest species. Additionally, the degradation of the soil as a consequence of deforestation hinders the establishment of new plants. Reforestation programs using nursery-produced seedlings have been implemented as a strategy for the recovery of forest areas. The production of such seedlings requires time and high production costs, and despite all care measures, the mortality rate of plants in Mexican reforestation programs is approximately 60–90% (Gómez-Romero et al. 2012). One of the species most used in Mexican reforestation and forest plantation programs is Pinus pseudostrobus, which is widely distributed in Mexico and Central America and is highly prized for its wood quality and productivity. It has also been successfully introduced in countries such as Brazil, New Zealand and South Africa (Mitchell et al. 2012; Cambrón-Sandoval et al. 2013; Zenni and Simberloff 2013). The use of PGPR, alone or in combination with ectomycorrhizal fungi, to produce high-quality seedlings are expected to decrease costs, nursery time and the use of fertilizers and pesticides. The aim of this study was to evaluate the growth promoter characteristics of rhizobacteria strains isolated from coniferous trees and to determine their effect on pine seedlings (P. pseudostrobus) in a nursery.

Materials and methods

Isolation of bacterial strains

Samples of rhizosphere soil were collected at a depth of 0–15 cm on Mount Tláloc, North of the Sierra Nevada in Central México, at an altitude between 3000 and 3800 m above sea level. Samples were sourced from the mycorhizosphere of oyamel (Abies religiosa), cedar (Cupressus lusitanica), and pine (Pinus hartwegii) trees. Additionally, samples were collected from the rhizosphere of 1-year-old Pinus montezumae trees grown in a nursery. The samples were transported to the laboratory in a cooler on ice. Bacterial strains were isolated using the dilution technique and plated on nutrient agar. Colonies showing abundant growth and with different morphologies were selected and purified by streaking onto nutrient agar. Pure bacterial cultures were stored at 4 °C until further use.

Auxin production

The bacterial strains were incubated in Luria–Bertani broth at 28 °C on a rotatory shaker (Model 2–16, Sigma) at 4×g for 180 rpm. Next, 3 mL of the bacterial cultures was transferred to 1.5 mL microcentrifuge tubes and centrifuged at 2655×g for 10 min. Then, 100 µL samples of the obtained supernatant were transferred to 96-well plates (microplates Costar 3591, Corning, NY); 200 µL of Salkowski reagent was added (2% v/v 0.5 M FeCl3 in 35% v/v perchloric acid), and the plates were incubated in darkness for 30 min for the development of color. Absorbance of the samples at 530 nm was measured with a spectrophotometer (Synergy 2 microplate reader, BioteK Instruments, Vermont, USA). The concentration of auxins was determined from a standard curve generated using 0–60 µg mL−1 of indole acetic acid.

Phosphate solubilization

The strains were cultured in tubes with Pikovskaya medium (10 g glucose, 5 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO4·7H2O, 0.2 g KCl, 0.5 g yeast extract, 0.002 g MnSO4, 0.002 g FeS04·7H2O, 1 L distilled water, pH 7)., incubated at 28 °C under agitation at 4×g for 180 rpm. After incubation, the bacterial cultures were centrifuged in Falcon tubes at 2655×g for 10 min. The supernatant was filtered through Whatman No. 1 filter paper, then 200 µL samples of each filtrate were transferred to 1.5 mL microcentrifuge tubes. Next, 100 µL of a solution of vanadate (0.25% w/v NH4VO3 in 35% v/v HNO3) and 100 µL of molybdate solution [5% w/v (NH4)6MO7O24 in distilled water] were added to each tube, which were mixed and incubated for 5 min. After this time, the samples were read at 420 nm. The amount of solubilized phosphorus was determined from a standard curve from 0 to 300 µg KH2PO4 mL−1 water.

Stimulation of ectomycorrhizal fungus growth

The ability of bacterial strains to stimulate the growth of ectomycorrhizal fungi was evaluated using strain CPEc1 of Suillus sp. from the collection of the Laboratory of Microbiology, Colegio de Postgraduados, Montecillo, Mexico. The strain was cultured in potato-dextrose agar (PDA) and incubated for 20 days, then 9-mm-diameter disks from the periphery of the colony was placed on PDA (pH 6.5) (Kataoka and Futai 2009). The bacterial strain was streaked in a straight line 2 cm from the disk of Suillus sp. The bacterial strains were cultured previously in nutrient broth for 7 days at 28 °C under constant agitation. As a control, a small sample of the medium without bacteria was streaked as above on a plate with a disk of the fungus. Three replicates per treatment were used. Petri dishes were incubated at 25 °C for 2 weeks, then the diameter of the fungal colony was measured.

Molecular identification

DNA was extracted from biomass equivalent to seven 24-h cultures of each bacterial strain with an EZ-10 Spin Column Bacterial DNA mini-prep kit (Bio Basic, Canada). The integrity of the DNA was evaluated by electrophoresis in a 1% agarose gel stained with Green-DNA Dye (Bio Basic) and visualized with a transilluminator.

A ~ 1500-bp fragment of the 16S rDNA was amplied for reliable identification to the level of genus and species using primers 27f (forward) 5′-AGA GTT TGA TCM TGG CTC AG-3′ and 1492r (reverse) 5′-CGG TTA CCT TGT TAC GAC TT-3′ and amplification reaction mixture containing 1× PCR Buffer, 2 mM dNTPs, 0.2 mM MgCl2, 10 µM of each primer and 0.05 U/µL DNA polymerase in a final volume of 25 µL. The thermocycler program included an initial denaturation for 5 min at 95 °C; 30 cycles of denaturation at 94 °C for 1 min, annealing at 57 °C for 1 min and extension at 72 °C for 1.5 min; with a final extension at 72 °C for 7 min. The amplicons were electrophoresed (100 V) in a 1.5% agarose gel and visualized with a transilluminator after staining with Green-DNA Dye (Bio Basic). The PCR products were then purified with an Agencourt AMPure XP kit (Beckman Coulter, USA) using the manufacturer’s instructions. The purified amplicons were sequenced at MACROGEN, Korea.

The sequences were edited with BioEdit software version 7.0.9.0 (Hall 1999) and Seaview version 4.0 (Galtier et al. 1996), and the phylogenetic trees were constructed with MEGA version 6.0 (Tamura et al. 2013). The sequences were used as queries in a search for homologous genes using the BLAST platform (http://blast.ncbi.nlm.nih.gov/Blast.cgi); the criteria reported by Rosselló-Mora and Amann (2001) were used to identify species (identity > 97%) and genus (identity of 95–96%), with coverage values higher than 85%. A phylogenetic analysis was performed in MEGA version 6 (Tamura et al. 2013) by mining sequences from GenBank (http://www.ncbi.nlm.nih.gov/genbank) obtained from a MegaBLAST analysis (Morgulis et al. 2008) to confirm the identity through the agreement in topology and the BLAST identity for each sequence of the strains. A phylogenetic tree was then constructed with using sequences of the strains and the mined sequences of microorganisms associated with plant growth promotion and a multiple alignment was done in ClustalX version 2.0 (Larkin et al. 2007) in MEGA6 (Tamura et al. 2013).

Nursery experiment

Seeds of P. pseudostrobus were submerged in water for 24 h; the water was changed every 6 h. The seeds were scarified with 30% H2O2 for 20 min under constant agitation, then rinsed with sterile distilled water to remove the H2O2. The seeds were then planted in germination trays using peat moss as substrate (previously sterilized for 6 h at 120 °C); two seeds were planted per well. The trays were covered with black plastic to accelerate germination and were maintained in a growth chamber at 28 °C for 28 days. Watering was performed with sterile distilled water, and every third day, a Captan solution was added (2 g L−1) to prevent the growth of pathogenic fungi. Two weeks after emergence, seedlings were transplanted to 140-mL tubes that were previously washed and disinfected with commercial chlorine and alcohol. The tubes were then filled with the substrate to 80% of capacity. The substrate consisted of a mixture of peat moss and sand (2:1), sterilized for 6 h at 120 °C. After the transplant, the seedlings were maintained in a nursery.

The bacterial strains were grown in nutrient broth (Merck) at 28 °C for 96 h in constant agitation, reaching 109 CFU mL−1. The culture was centrifuged at 2655×g for 10 min; the supernatant was removed, and the bacterial pellet was resuspended in sterile distilled water. The seedlings were inoculated with 2 mL of this suspension at the base of the seedling stem. Inoculum was added twice to ensure the persistence of the bacterium in each treatment: the first, 2 weeks after the transplant; and the second, 3 months after the first inoculum. The plants were watered with distilled water.

The treatments consisted of 10 rhizobacterial strains and an unfertilized and noninoculated control, which were established in a randomized block experimental design with 10 replicates. The plants were harvested 150 days after the establishment of the experiment. The variables evaluated were the height, diameter and dry mass of the shoots and roots. The plant material was oven-dried (Jouan Model EUSS ELTS SN) at 70 °C until reaching constant mass. Total nitrogen and phosphorus in the plant was determined by the micro-Kjeldahl and molybdo-vanadate methods, respectively. The data were analyzed using SAS version 9 (SAS Institute Inc 1999) for Windows (Microsoft, Redmond, WA, USA) to obtain the analysis of variance (ANOVA) and means comparison testing using the least significant difference (LSD) with α = 0.05.

Results

Isolation and characterization

Ten strains were isolated from the mycorrhizosphere of C. lusitanica, A. religiosa, P. hartwegii, and P. montezumae (Table 1). All strains produced 1.6 µg to 76.4 µg mL−1 indole acetic acid. Strains CPT5, CPT6, CPT7, CPT8, and CPT9 produced more than 33 µg mL−1 IAA. Seven strains solubilized phosphorous; strain CPT6 solubilized 433 µg mL−1 phosphorous.

Table 1 Molecular identification and concentrations of IAA produced and phosphorous solubilized (Sol.) by the isolated rhizobacterial strains
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Induction of mycelial growth of Suillus sp. in vitro

Bacterial strain CPT8 induced mycelial growth of Suillus sp. in vitro by 18.8% compared with the control mycelia. Strains CPT5 and CPT6 inhibited growth of Suillus sp. by 6 and 4%, respectively. The remaining strains did not significantly affect fungal growth.

Bioassay of bacterial inoculation in P. pseudostrobus

Some of the strains had significant beneficial effects on the variables evaluated (Table 2). Strain CPT2 increased the height of P. pseudostrobus by 68.9% compared to that of the control. Strains CPT4, CPT6 and CPT9 increased the dry mass of the shoots by 20.4, 15.2 and 24.2%, respectively (Fig. 1). Other strains inhibited plant growth. For example, plants treated with strain COT7 had the lowest values for stem diameter (1.38 mm), shoot dry mass (0.24 g), and total biomass (0.3437 g).

Table 2 Height, diameter and P and N content for P. pseudostrobus seedlings 150 days after inoculation with different rhizobacterial strains
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Fig. 1
figure 1

Effect of different rhizobacteria strains in P. pseudostrobus on dry mass of a shoots and b roots 150 days after inoculation

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Identification of strains by molecular biology

The 10 strains were identified as belonging to the following species: C. basilensis (CPT1, CPT2, CPT3), R. qingshengii CPT4, Pseudomonas sp. CPT5, Pseudomonas gessardii CPT6, P. gessardii CPT7, S. rhizophila CPT8, Rhodococcus erythropolis CPT9 and Cohnella sp. CPT10. Of the group of strains isolated, those belonging to the genus Pseudomonas came from the ectomycorrhizosphere of P. hartwegii and A. religiosa. The nucleotide sequences for the strains were submitted to GenBank database and assigned accession numbers KU195255 (CPT1), KU195256 (CPT2), KU195257 (CPT3), KU195258 (CPT4), KU195259 (CPT5), KU195260 (CPT6), KU195261 (CPT7), KU195262 (CPT8), KU195263 (CPT9), and KU195264 (CPT10).

In the phylogenetic analysis of the genetically identified strains using sequences from bacterial strains related to these species and the ectomycorrhizosphere from which they were isolated (Fig. 2), the sequences clustered in the taxonomic groups Beta-proteobacteria, Gamma-proteobacteria and Actinobacteria.

Fig. 2
figure 2

Phylogeny of the identified strains

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Discussion

The results obtained in this study showed that bacteria of the genera Rhodococcus and Pseudomonas act as growth promoters in P. pseudostrobus. However, the increase in the production of biomass did not always depend on the production of IAA or on phosphorous solubilization by the bacteria. The effect of the secondary metabolites produced by the bacteria might depends on the affinity between organisms. Pseudomonas aeruginosa showed strong chemotaxis in the presence of the root exudates of Pinus roxburghii (Singh et al. 2010). Pseudomonas fluorescens is capable of producing auxins and dissolving calcium phosphate, which allows it to properly establish itself in the rhizosphere of Pinus pinea (García et al. 2004; Karnwal 2009). P. fluorescens has been widely studied because of its growth-promoting capacity and has been found in the mycosphere of ectomycorrhizal fungi (ECMF) such as Lactarius sp. and Rhizopogon sp. (Preston 2004; Aspray et al. 2006; Frey-Klett et al. 2007). The three strains of Pseudomonas tested in the experiment increased the shoot and root biomass of P. pseudostrobus; however, they did not enhance growth of the ectomycorrhizal fungi. R. qingshengii and R. erythropolis also had a positive effect on biomass production. However, R. qingshengii promoted growth of the ECMF; therefore, it can promote mycorrhizal colonization. Few studies have been performed with bacteria from the genus Rhodococcus, although found in the mycorrhizosphere of ECMF such as Lactarius rufus (Poole et al. 2001). Although R. erythropolis has not been reported as a species that promotes mycorrhizal colonization, it has potential in areas such as bioremediation as a sulfur-removing agent (Naito et al. 2001; Gogotov and Khodakov 2008; Cumming et al. 2015). It is also capable of using phenol and pyrocatechol, among other substrates, for its growth (Čejková et al. 2005) and can degrade paraffin (Zhukov et al. 2007) and diesel (Zhang et al. 2007). Although C. basilensis has not been reported as PGPR, some species of Cupriavidus form associations with legumes and are resistant to heavy metals (Estrada de los Santos et al. 2012). C. basilensis has been reported for its capacity to degrade furanic compounds (Wierckx et al. 2011). S. rhizophila also enhanced biomass and root growth. So dual inoculation with these two strains could induce mycorrhizal formation in plant species of interest for forestry, such as P. pseudostrobus. Dual inoculation (bacterial-ECMF) has been widely studied with positive, neutral or negative responses depending on the affinity between microorganisms (Bonfante and Anca 2009; Hrynkiewicz et al. 2010; Wu et al. 2012).

The PGPR promote plant growth by increasing nutrient availability (Pii et al. 2015). Nutrient solubilization and mineralization can be biological or biochemical through the production and release of organic acids, such as citric, butyric, acetic and fumaric acids, phenolic compounds, and siderophores (Ouahmane et al. 2009). Organic acids produced by rhizobacteria acidifies the medium (pH 7 to pH 4.2), allowing the solubilization of phosphorous (Song et al. 2008). Similarly, strain CPT6 in the present study acidified the pH of the medium compared to the control (pH 8 to 6). Paenibacillus polymyxa decreases the mortality of Pinus contorta var. Latifolia seedlings and increases the concentration of N in seedlings (Anand et al. 2013). Strains of the genus Pseudomonas increase the height and dry mass of Pinus taeda seedlings (Brunetta et al. 2010) There are reports in which C. basilensis is capable of solubilizing phosphorous (Qian et al. 2010), but the strains in this experiment did not show this capacity. Accordingly, the isolated strains are capable of solubilizing considerable amounts of phosphorous, which remains available for the plant. In soils in which phosphorous is immobilized, this mechanism is a viable solution for decreasing fertilizer use (White and Metcalf 2007).

The use of PGPR to produce seedlings of forest species for the reforestation of altered areas still requires more research because little information is available. The present studies showed favorable increases in biomass. The use of dual inoculants is a strategy to decrease the application of fertilizers and pesticides. This strategy would allow the establishment of endophyte communities in the rhizosphere that promote positive effects on plant physiology, such as stimulation of photosynthesis, release of root exudates, and production of growth factors (Fuentes-Ramírez and Cabellero-Mellado 2005; Barka et al. 2006).

Despite the benefits PGPR offer, potential consequences to human health need to be assessed (Fuentes-Ramírez and Cabellero-Mellado 2005). For example, R. erythropolis has been reported in six cases of infection in immunocompromised patients (Park et al. 2011). Therefore, bacterial inoculants should be applied in the field with great care and with appropriate quality control.

Conclusions

The bacterial strain that best stimulated the growth of P. pseudostrobus increased shoot dry mass by 24.2% over the control and by dry mass of roots by 33.9%. A more profuse root system leads to better nutrient capture from the soil. The strains were molecularly identified to belong to the genera Pseudomonas, Rhodocccocus, Cupriavidus, Stenotrophomonas and Cohnella, which have been found in association with hyphae of ectomycorrhizal fungi; thus, these strains may be more efficient growth promoter after a dual inoculation with an ectomycorrhizal fungus. However, studies evaluating the effect of mycorrhizal colonization in conjunction with plant-growth promoting bacteria are still needed.