Introduction

Plant growth-promoting microorganisms (PGPMs) correspond to any microorganism that, through its direct or indirect action, enhances plant growth (Altomare and Tringovska 2011). These microorganisms include nitrogen (N)-fixing bacteria, phosphorus (P)-solubilizing microorganisms and microorganisms synthesizing plant hormones (Altomare and Tringovska 2011). N-fixing and P-solubilizing microorganisms improve plant growth by increasing the N or P availability in the root zone, thus reducing the energy expenditure of roots for their uptake. In the group of microorganisms capable of synthesizing plant hormones, several soil bacteria and fungi have been identified to release auxins, gibberellic acid, and/or cytokinins (Sofo et al. 2011), becoming the active ingredient in a large number of biofertilizers used worldwide (Mitter et al. 2021). Recently, the production of the auxin indole-3-acetic acid (IAA) along with the activity of 1-aminocyclopropnae-carboxylate (ACC) deaminase were proposed as the main traits to be identified in novel PGPMs (Ali et al. 2022).

Several soil yeast strains have been reported to synthesize plant hormones, but in comparison to biofertilizers based on bacteria or filamentous fungi, very few based on yeast are commercially available (Hernández-Fernández et al. 2021). Yeasts are unicellular fungi that perform sexual and asexual reproduction through budding or cell fission (Botha 2011). Soil yeasts in genera such as Aureobasidium or Coniosporium present P-solubilizing capacity, while yeasts in genera such as Candida, Cryptococcus, Rhodotorula, Sporobolomyces, or Williopsis have been reported to produce and release IAA (Botha 2011). Yeasts synthesizing IAA have been reported as effective root growth promoters in a variety of soil conditions (Ali et al. 2009; Hayat et al. 2010 and references therein; Yuan et al. 2011) due to the induction of lateral root initiation. However, the positive effects of IAA in the medium are observed when the concentration of this hormone is within a certain range; the opposite effect occurs when its concentration is above the suitable range, depending on the crop (Gravel et al. 2007).

Recently, the identification of soil microorganisms capable of reducing plant ethylene synthesis has gained substantial attention among researchers worldwide. Ethylene is a gaseous hormone synthesized in plants, but under stress conditions, its concentration rapidly increases, triggering a series of responses, including cell division detention, interruption of cell expansion, leaf growth retardation, and leaf senescence, among others (Dubois et al. 2018). Certain microorganisms present the capacity to reduce plant ethylene synthesis due to the enzyme ACC deaminase, which cleaves ACC into ammonia and α-ketobutyrate (Polko and Kieber 2019). ACC is an ethylene precursor; therefore, the activity of ACC deaminase reduces the ethylene concentration in plant tissue, resulting in growth promotion. To date, more than 40 rhizobacteria, including Azospirillum brasilense, Bacillus subtilis, Burkholderia phytofirmans and Pseudomonas aeruginosa, have been reported to present ACC deaminase activity (Raghuwanshi and Prasad 2018), while only two soil yeasts, Candida tropicalis (Amprayn et al. 2012) and Meyerozyma guilliermondii (Aban et al. 2017), have been identified with ACC deaminase activity. The beneficial effects of reduced ACC in plants occur under normal and stress conditions, as shown by Martínez-Andujar et al. (2016), who worked with different tomato rootstocks and concluded that lower concentrations of ACC in the roots promoted vigorous growth of the whole plant.

Compared to bacteria or filamentous fungi, yeasts have the advantage of presenting a more stable genome and are easier to culture (Chen et al. 2022). Thus, identifying soil yeasts with ACC deaminase activity will be beneficial for the future production of biofertilizers. Therefore, the aim of the present study was to identify soil yeast strains isolated from soils in Chile that present plant growth-promoting activity, either by synthesizing and releasing IAA or showing ACC deaminase activity.

Materials and methods

Twenty-three yeast strains (Table 1) isolated from soils from various locations in Chile were evaluated to determine their capacity to synthesize IAA or to present ACC deaminase activity. All strains are part of the collection held at the Laboratorio de Microbiología y Genética de Levaduras, Pontificia Universidad Católica de Chile (PUC) and were sequenced (Macrogen, South Korea) to confirm their identification.

Table 1 List of soil yeasts evaluated in the present study. All the strains are catalogued in the yeast collection (YC) held at Pontificia Universidad Católica de Chile (PUC) and were originally collected from various locations and soil types in Chile
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IAA synthesis quantification

Yeasts were individually cultured in yeast extract-peptone-dextrose (YPD) liquid medium (1% yeast extract, 2% peptone and 2% glucose) with and without the addition of 0.1% (m/v) L-tryptophane. The culture was maintained for one week under continuous agitation at 150 rpm under dark conditions and an air temperature of 28 °C. Cumulative IAA production at the end of the week was quantified following the Salkowski method (Ehmann 1977). For this, 0.1 mL of the supernatant was mixed with 0.1 mL of Salkowski’s reagent (2 mL Fe2Cl3 + 98 mL 35% HClO4) in a microplate using triplicates per strain. Then, the plates were incubated for 30 min in the dark under ambient temperature, and the concentration of IAA was determined by spectrometry at 530 nm.

ACC deaminase activity determination

The activity of the enzyme ACC deaminase was first determined qualitatively. For this, each strain was individually cultured in YPD medium for two days. Then, 50 μL of the supernatant was added to test tubes along with yeast nitrogen base (YNB) medium with no nitrogen source other than 3 mM ACC. The tubes were allowed to rest for two weeks in an incubator set at 150 rpm and 28 °C. The development of turbidity in the medium was analyzed by spectrophotometry at 630 nm as the indicator of successful growth.

Once the strains with ACC deaminase activity were identified, the enzymatic activity was quantified by measuring the production of α-ketobutyrate, following the method proposed by Torbaghan et al. (2017). For this, 1 mL of the culture obtained from the previous stage was centrifuged for 5 min at 3000×g, and the supernatant was discarded. Then, the pellet was suspended in 1 mL of 0.1 M Tris-HCl at pH 7.6 and centrifuged for 5 min at 16,000×g, and the supernatant was discarded again, with pellet resuspension in 0.2 mL of 0.1 M Tris-HCl at pH 8.5. Then, 30 μL of toluene was added, and 0.2 mL of this solution was mixed with 20 μL of 0.5 M ACC. The solution was incubated for 15 min at 30 °C, and after this, 1 mL of 0.56 M HCl was added and the mix was centrifuged for 5 min at 16,000×g. One mL of the supernatant was sampled and added to 0.8 mL of 0.56 M HCl. Samples were placed in a vortex where 0.3 mL of 2,4-dinitrophenilhydrazine was added, homogenized and incubated for 30 min at 30 °C. Finally, 2 mL of 2 M NaOH was added, and the concentration of α-ketobutyrate was determined by spectrophotometry at 540 nm.

In vitro inoculation of tomato seeds with strains synthesizing IAA or presenting ACC deaminase activity

Each strain with the capacity to synthesize IAA or to deaminate ACC was selected and used in a coculture with tomato seeds to determine their effect on plant growth. Five seeds of tomato cv. Attiya (Rijk Zwaan) were placed in Petri dishes with Murashige and Skoog (MS) medium supplemented with 1% sucrose, 1.5% agar and 0.05% 2-(N-morpholino) ethenesulfonic acid (MES). Seeds were disinfected in a 1% v/v sodium hypochlorite solution for 15 min and then rinsed with sterile distilled water. Four replicates of each strain plus a control with no yeast were placed in a dark chamber at 28 °C for 48 hours, after which the dishes were inoculated with 50 μL of a saturated yeast culture. Using a glass rake, the yeast culture was distributed in Petri dishes and incubated for 5 days under 25 °C and a 16 h photoperiod. At the end of the five days, the number of lateral roots and total root volume were determined using WinRhizo software.

In vivo experiment

From the previous experiment, two yeast strains were selected to evaluate their effect in vivo on plant growth: one strain synthesizing IAA (Metschinikowia sp. YCPUC89) and one with ACC deaminase activity (S. aeria YCPUC79). The growth of both strains in the nutrient solution was checked prior to the start of the experiment. A growth chamber experiment was set with four treatments: control (no yeast addition), IAA (addition of an IAA-synthesizing strain), ACC (addition of a strain with ACC deaminase activity), and IAA + ACC (addition of both strains). The treatments were applied adding the corresponding strain to the nutrient solution in 3 L plastic containers each containing two thirty-day-old tomato plants. The composition of the nutrient solution was as follows: 7 mM N, 0.5 mM P, 3 mM potassium (K), 2 mM calcium (Ca), 1 mM magnesium (Mg) and 1 mM sulfur (S). The solution was mixed using reverse osmosis water, and the complete solution was replaced twice per week. Four replicates of each treatment were randomly distributed in a growth chamber with a photoperiod of 16 h using LED lamps (model B200, Valoya) providing 350 μmol PAR m−2 s−1. The air temperature and relative humidity were kept at 25 ± 1 °C and 70 ± 5%, respectively. Plants were grown for 21 days, and regular measurements of root ethylene production were performed using an ethylene analyzer set at a 10 mL min−1 flow rate (model F-900, Felix Instruments). On the final day of the experiment, before harvest, the leaf chlorophyll content and CO2 assimilation rate were measured using a chlorophyll meter (model MC-100, Apogee instruments) and an infrared gas analyzer (model LI-6400XT, Licor Bioscience), respectively. The conditions within the cuvette were set at a 300 μmol s−1 air flow rate, 800 μmol PAR m−2 s−1, CO2 concentration of 400 ppm and ambient relative humidity. Then, plants were harvested, split into roots and shoots, and fresh weight was recorded. Plants were dried in an oven at 55 °C for 72 h, and dry weight (DW) was recorded. Root DW was used to normalize the ethylene production rate measured on the last day of the experiment.

Statistical analysis

Differences in IAA synthesis, ACC deaminase activity, number of lateral roots and root volume among the strains were assessed by one-way ANOVA followed by mean separation using Fisher’s LSD test at 5% significance level. Differences in root ethylene synthesis, leaf chlorophyll content, leaf gas exchange measurements, and plant fresh and dry weight among the treatments were evaluated by ANOVA and mean separation by LSD test. All statistical analyses were performed using Statgraphics Centurion XVI.I software (StartPointTechnologies, Warrenton, USA).

Results

IAA synthesis and ACC deaminase activity identification in the yeast strains

The cumulative IAA synthesis of the strains after one week of culture was within the range of 0.8 to 3.3 μg IAA mL−1 when no tryptophan was added to the medium. Higher concentrations were obtained in sixteen of the strains when tryptophan was added, while five strains produced the same amount and two reduced their production (Fig. 1). Rhodotorula babjevae YCPUC42 was the strain with the highest production, with over 3.0 μg IAA mL−1, while two strains of P. membranifaciens, YCPUC66 and YCPUC144, presented the lowest IAA synthesis with values below 1.0 μg IAA mL−1.

Fig. 1
figure 1

Indole-3-acetic acid (IAA) synthesis evaluated in the 23 soil yeast strains. Each column represents the mean ± s.e. of three replicates. Different letters on top of the columns denote significant differences among yeast strains (p < 0.05) after LSD test. Asterisks on top of the columns denote significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.0001) within the same strain with (black columns) or without (grey columns) the addition of tryptophan

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Only two strains of S. aeria, YCPUC75 and YCPUC79, presented ACC deaminase activity, with production rates of 12.77 ± 0.14 and 14.86 ± 0.12 μmol α-ketobutyrate min−1 L−1, respectively.

In vitro effects of soil yeast strains on tomato seedlings morphological parameters

Germination of tomato seeds in Petri dishes inoculated with the yeast strains resulted in significant differences in root volume (p < 0.0001) and the number of lateral roots (p < 0.0001), with some strains producing positive effects on both traits and even some strains result in negative effects in comparison to the control (Fig. 2).

Fig. 2
figure 2

Tomato seedlings after five days of coculture with the yeast strains in MS medium supplemented with 1% sucrose, 1.5% agar and 0.05% MES. A: Control; B: Wickerhamonyces onychis YCPUC87; C: Metschinikowia sp. YCPUC89; D: Solicoccozyma aeria YCPUC78; E: Debaryomyces hansenii YCPUC137; F: Debaryomyces hansenii YCPUC136; G: Pichia membranifaciens YCPUC144; H: Pichia kluyveri YCPUC83; I: Suhomyces kilbournensis YCPUC113; J: Suhomyces kilbournensis YCPUC101; K: Rhodotorula dairenensis YCPUC35; L: Pichia membranifaciens YCPUC66; M: Solicoccozyma aeria YCPUC79; N: Rhodotorula babjevae YCPUC42; O: Solicoccozyma aeria YCPUC77; P: Solyicoccozyma. aeria YCPUC75; Q: Torulaspora delbrueckii YCPUC10; R: Wickerhanomyces onychis YCPUC85; S: Torulaspora delbrueckii YCPUC174; and, T: Saccharomyces bayanus YCPUC166

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The highest value for root volume was achieved with R. dairenensis YCPUC35, reaching 58.94 ± 5.06 mm3 plant−1 vs. 32.82 ± 2.14 mm3 plant−1 in the control (Fig. 3A). The number of lateral roots increased from 9.52 ± 0.92 in the control to 21.64 ± 1.17 with S. aeria YCPUC77 and R. babjevae YCPUC42 (Fig. 3B).

Fig. 3
figure 3

Root parameters after the coculture of tomato seedlings with each yeast strain. A: root volume; B: number of lateral roots. Each column denotes the mean ± s.e. of four replicates. The control treatment is depicted in the black column. Different letters denote significant differences (p < 0.05) after LSD test

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Effect of yeast on plant growth and ethylene synthesis

The leaf chlorophyll content was higher in all treatments (p < 0.0001) in comparison to the control, while the leaf CO2 assimilation rate was higher in plants exposed to Metschinikowia sp. YCPUC89 (IAA synthesizing strain) and S. aeria YCPUC79 (yeast with ACC deaminase activity) treatments (p < 0.0188) (Table 2). Solicoccozyma aeria YCPUC79 (ACC treatment) increased shoot biomass in comparison to the control (p < 0.0001) treatment (no inoculation) but did not affect root dry biomass. Nevertheless, Metschinikowia sp. YCPUC89 and Metschinikowia sp. YCPUC89 + S. aeria YCPUC79 reduced the root biomass (p < 0.0022) in comparison to the control.

Table 2 Leaf chlorophyll content and CO2 assimilation rate, shoot and root dry weight (DW) and root ethylene synthesis measured 21 days after transplant on tomato plants exposed to S. aeria YCPU79 (strain with ACC deaminase activity), Metschinikowia sp. YCPUC89 (IAA synthesizing strain), the combination of both strains plus the control (no inoculation). Values represent the mean ± s.e. of four replicates. Different letters in the same column denote significant differences (p < 0.05) after LSD test
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The ethylene concentration rapidly increased three days after transplant in all treatments but started decreasing six days later (Fig. 4). The treatments presenting solely S. aeria YCPUC79 showed the lowest ethylene concentration throughout the experiment.

Fig. 4
figure 4

Evolution of the ethylene concentration in the root zone during the 21 days of the experiment in the growth chamber. Control: non-inoculated; IAA: inoculated with Metschinikowia sp. YCPUC89 (IAA synthesizing strain); ACC: inoculated with S. aeria YCPUC79 (strain with ACC deaminase activity); and, IAA + ACC: inoculation with the Metschinikowia sp. YCPUC89 and S. aeria YCPUC79. Symbols represent the mean ± s.e. of four replicates

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Discussion

All the yeast strains evaluated in the present study synthesized and released IAA into their surrounding environment. The synthesis of this hormone is required for the adhesion of yeast to host plants (Prusty et al. 2004). Thus, biofertilizers including yeasts in their mix should ensure successful colonization of the rhizosphere. However, root growth can be inhibited by high concentrations of IAA, as shown by Muday et al. (1995), who reported that root growth in tomato seedlings decreased with IAA concentrations higher than 0.01 μM (equivalent to 1.75 ng mL−1). Our results indicate that two strains of T. delbrueckii, YCPUC10 and YCPUC174, one of S. bayanus, YCPUC166, and one of W. onychis, YCPUC85, restricted root and plant growth, but these strains did not have the highest IAA synthesis. Thus, the growth restriction observed with these strains is not mediated by high concentrations of IAA. In fact, the strain with the highest IAA synthesis was R. babjevae YCPUC42, which resulted in a similar number of lateral roots to the strain S. aeria YCPUC77, which synthesized only 33% of that amount of IAA. Volatile compounds synthesized by soil microorganisms have been identified to have either positive or negative effects on plant growth (Delaplace et al. 2015); nonetheless, those compounds were not studied in the present research.

On the other hand, ACC deaminase has been identified as a promising feature for the development of new biostimulants (Ali et al. 2022; Sangiorgio et al. 2020). In a study on tomato where eight species of Bacillus were evaluated, significant increases in plant biomass accumulation were observed only with those genotypes showing ACC deaminase activity (Kalam et al. 2020). Similar results have been reported in various crops, including Zea mays (Zarei et al. 2020), Triticum aestivum (Zafar-ul-Hye et al. 2019), Oryza sativa (Sarkar et al. 2018), and Phaseolus vulgaris (Gupta and Pandey 2019). To the best of our knowledge, this is the first report of ACC deaminase activity in S. aeria, which has been previously reported only in one other soil yeast, Candida tropicalis, or in soil bacterium like Pseudomonas putida S-313 (Amprayn et al. 2012). Solicoccozyma aeria has been characterized as a soil yeast from arid climates (Yurkov 2018), but the collection held at Pontificia Universidad Católica de Chile includes strains collected from semiarid through temperate rainy climates. Within this collection, two strains of S. aeria present ACC deaminase activity, one from the semiarid region in northern Chile (YCPUC75) and one from southern Chile (YCPUC79). The advantage of S. aeria in comparison to C. tropicalis may lie in the fact that yeasts in the Candida genus have been isolated from soils in temperate humid environments (Vadkertiová et al. 2017); therefore, S. aeria is capable of adapting to different types of soils under various climatic conditions.

The combination of ACC deaminase activity and IAA synthesis has been previously reported as a successful trait in soil microorganisms promoting growth in various crops, such as tomato (Khan et al. 2016; Kang et al. 2019), rice (Amprayn et al. 2012; Bhattacharjee et al. 2012), Capsicum annuum (Choudhury et al. 2021), and Brassica napus (Madhaiyan et al. 2007). In the present study, the addition of S. aeria YCPUC79 to the nutrient solution increased shoot growth in tomato by 26% in comparison to the control, with a 40% reduction in root ethylene synthesis under normal conditions. Reducing root ethylene synthesis is a promising tool for crop adaptation to stressful conditions since it alleviates negative effects of ethylene, leading to organ senescence and abscission (Raghuwanshi and Prasad 2018). For instance, a 40% reduction in ethylene synthesis in wheat seedlings exposed to salinity stress was reported with Trichoderma longibrachiatum (Zhang et al. 2019), and a 30% reduction in ethylene synthesis was reported in transgenic tomato plants expressing the bacterial gene ACC deaminase under waterlogging conditions (Grichko and Glick 2001).